Methods for sequential DNA amplification and sequencing

ABSTRACT

Homogenous detection during or following PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled primers and probes, improves reproducibility and quantification. Low-temperature homogeneous detection during or following non-symmetric PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled mismatch-tolerant probes permits analysis of complex targets. Sequencing sample preparation methods following LATE-PCR amplifications reduce complexity and permit “single-tube” processing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/292,038 filed Nov. 10, 2008, now abandoned, which is a division ofU.S. patent application Ser. No. 11/252,433 filed Oct. 17, 2005, nowU.S. Pat. No. 7,632,642, which claims priority to U.S. ProvisionalApplication 60/619,654 filed Oct. 18, 2004, each of which are hereinincorporated by reference in their entireties.

TECHNICAL FIELD

This invention relates to nucleic acid amplification reactions,including amplifications utilizing the polymerase chain reaction, andassays utilizing such reactions in combination with sequencing andhybridization probe detection methods.

BACKGROUND

Nucleic acid amplification techniques and assays are well known. Someamplification reactions are isothermal, such as nucleic acid sequencebased amplification (NASBA). Others employ thermal cycling, such as thepolymerase chain reaction (PCR). Preferred amplification assaysemploying fluorescence detection of amplified product are homogeneous,that is, they do not require the physical separation of reagents topermit detection (for example, separation of bound probes from unboundprobes) and can be performed in a single closed vessel. Such assays maybe end-point, wherein product is detected after amplification, or theymay be real-time, wherein product is detected as amplification proceeds.

Nucleic acid amplification and assays employing PCR are described, forexample, in U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,965,188, and,generally, PCR PROTOCOLS, a guide to Methods and Applications, Innis etal. eds., Academic Press (San Diego, Calif. (USA) 1990). Homogeneous PCRassays, including real-time assays, in which amplified product isdetected during some or all of the PCR cycles as the reaction proceedsare described, for example, in U.S. Pat. Nos. 5,994,056, 5,487,972,5,925,517 and 6,150,097.

PCR amplification reactions generally are designed to be symmetric, thatis, to make double-stranded amplicons exponentially by utilizing forwardprimer and reverse primer in equimolar concentrations and equal meltingtemperatures (T_(m)'s). A technique that has found limited use formaking single-stranded DNA directly in a PCR reaction is “asymmetricPCR.” Gyllensten and Erlich, “Generation of Single-Stranded DNA by thePolymerase Chain Reaction and Its Application to Direct Sequencing ofthe HLA-DQA Locus,” Proc. Natl. Acad. Sci. (USA) 85: 7652-7656 (1988);and U.S. Pat. No. 5,066,584. Asymmetric PCR is a non-symmetric PCRamplification method that differs from symmetric PCR in that one of theprimers is diluted fivefold to one hundredfold so as to be present inlimiting amount of 1-20 percent of the concentration of the otherprimer. As a consequence, the amplification consists of an exponentialphase in which both primers are extended, generating double-strandedproduct, or amplicon, followed by a linear amplification in which onlyone primer remains, generating single-stranded amplicon.

More recently we have developed a non-symmetric PCR amplification methodknown as “Linear-After-The-Exponential” PCR or, for short, “LATE-PCR.”LATE-PCR is a non-symmetric PCR amplification consisting of anexponential phase in which both primers are annealed and extendedfollowed by a linear phase after exhaustion of the Limiting Primer, whenonly the Excess Primer is annealed and extended. See Sanchez et al.(2004) Proc. Natl. Acad. Sci. (USA) 101: 1933-1938, publishedinternational patent application WO 03/054233 (3 Jul. 2003), and Pierceet al. (2005) Proc. Natl. Acad. Sci (USA) 102: 8609-8614, all of whichare incorporated herein by reference in their entirety.

A convenient and inexpensive method for monitoring double-strandedamplicon production in a PCR amplification is to use a dye thatfluoresces upon intercalating into or otherwise interacting withdouble-stranded DNA, such as SYBR Green I or SYBR Gold. See, forexample, U.S. Pat. No. 5,994,056. Melting temperature analysis ofamplicons performed either in real time during a PCR amplification orperformed after amplification is used for product identification. Oneproblem with utilizing such melting temperature analysis is that dyefluorescence is a function of amplicon size. Another problem is thatdyes redistribute from amplification products, or amplicons, having lowmelting temperatures to amplicons having higher melting temperaturesduring analysis, thereby distorting results. Two approaches to solvethese problems have been advanced. One approach, G quenching, imposessevere restrictions on primer design and causes large backgroundfluorescence (Crockett A O, Wittwer C T. “Fluorescein-LabeledOligonucleotides for Real-Time PCR: Using the Inherent Quenching ofDeoxyguanosine Nucleotides” Anal. Biochem. 290:89-97 (2001)). The other,replacing SYBR dyes with LC Green dye, yields very small percentage ofsignal for sequences not present in abundance and requires highlyspecialized software and hardware (Wittwer et al. High-ResolutionGenotyping by Amplicon Melting Analysis Using LCGreen,” Clin. Chem.49:853-860 (2003).

Fluorescent-labeled probes are used in homogeneous nucleic acidamplification assays, including PCR assays, to measure the accumulationof desired amplicon, either in real time or by end-point analysis.Several available types of probes are significantlyallele-discriminating as compared to linear single-stranded probes.Real-time probes include dual-labeled linear probes that are cleaved by5′-to-3′ exonuclease activity of DNA polymerase during the extensionstep of a PCR cycle (see U.S. Pat. Nos. 5,210,015, 5,487,972 and5,538,848); molecular beacon probes (see U.S. Pat. Nos. 5,925,517,6,103,476 and 6,365,729); minor groove binding probes (see Afonina etal. “Minor Groove Binder-Conjugated DNA Probes for Quantitative DNADetection by Hybridization-Triggered Fluorescence,” Biotechniques 32:946-949 (2002)); linear probe pairs that FRET when hybridized adjacentlyon a target strand; quenched double-stranded linear probes for which atarget competes to hybridize to the labeled probe strand (see Li, Q. etal. (2002), Nucl. Acid. Res. 30: e5); and so-called “light-up” probes,which are peptide nucleic acid (PNA) oligomers linked to an asymmetriccyanine dye that fluoresces when the probe binds to target to form adouble-stranded region. For LATE-PCR we have utilized low-temperatureallele-discriminating probes, such as low temperature molecular beaconprobes (See WO 03/045233). Labeled oligonucleotide probes may beattached to primers by linkers such that during amplification the probesare not copied but are free to hybridize to a target sequence resultingfrom extension of the primer. Examples are Scorpions®, primers to whichare attached molecular beacon probes, and Anglers®, primers to which areattached fluorophore-labeled linear probes. Lee, M. A. et al. (2002),Analytica Clinica Acta 457: 61:70; Whitcombe, D. et al. (1999), NatureBiotechnology 17: 804-807. The probe portion of such compositestructures, which carries the fluorescent label, hybridizes separatelyfrom the primer portion. They are, thus, labeled probes and not labeledprimers, as those terms are used herein. Target-specific probes lack thecapacity to monitor total production of double-stranded products,however.

Certain probes are mismatch-tolerant. Mismatch-tolerant probes hybridizewith and generate detectable signal for more than one target sequence ata detection temperature in an assay, and various hybrids so formed willhave different melting points. Linear, or random coil, single-strandedprobes are generally mismatch tolerant. Examples of such probes arelinear probes with an internal fluorescent moiety whose level offluorescence increases upon hybridization to one or another targetstrand; fluorescently labeled linear probes used in combination withSYBR Gold and SYBR Green I dyes, such that fluorescence of the labeloccurs by FRET from the dye when the probe hybridizes to one or anothertarget (see U.S. patent publication US 2002/0119450, 28 Aug. 2002),so-called “sloppy beacons”, and variations of linear probe pairs thatFRET (see U.S. Pat. No. 6,472,156).

Utilizing multiple probes that each bind only to one possible targetamplicon generated in an amplification reaction creates a problem foranalyzing complicated reaction mixtures or for detecting one or a fewtargets from among numerous possible targets. Available fluorescencedetection permits resolution of a limited number of fluorophores,generally no more than eight. Limited multiplexing is possible, forexample, by designing a different allele-discriminating molecular beaconprobe for each target and labeling each probe differentially. (See, forexample, Tyagi et al. (2000) Nature Biotechnology 18: 1191-1196).Mixtures of allele-discriminating probes, each comprising aliquots ofmultiple colors, extends the number of probe signatures and works wellif only one of many (at least up to 56) targets is actually present, butit encounters ambiguous results if more than one target is present.

There are many situations that involve complex targets or one among manypossible targets. Several schemes have been developed or proposed forsuch situations, but all have serious drawbacks and limitations. Tyagiet al. published international patent application WO 01/31062, havedescribed a technique sometimes referred to as “sloppy beacons,” whichare molecular beacon probes that have long loop sequences, renderingthem mismatch tolerant and able to bind to some extent to multipletargets at the annealing temperature of a PCR amplification reaction.Such probes suffer from poor reaction kinetics against mismatchedtargets and are likely to remain hybridized to perfectly matched targetsat the extension temperature of a PCR amplification and be cleaved byTaq DNA polymerase. Further, only an indirect indication of meltingtemperatures of probe-target hybrids under the assay conditions isobtained, and that assumes equilibrium has been achieved. Real-timemultiplexing in symmetric PCR amplifications with FRET probes has beendescribed. In order not to interfere with amplification, the meltingtemperatures of all probe-target hybrids are constrained to be in thenarrow temperature range between the primer annealing temperature andthe primer extension temperature. Also, that scheme is not quantitative.Post-amplification multiprobe assays employing FRET probes of differentcolors have been disclosed Wittwer et al., “Real-Time Multiplex PCRAssays, Methods” 25:430-442 (2001). The reaction mixture following asymmetric PCR amplification is rapidly chilled, then slowly heated todetermine melting curves for the various fluorophores present. Thisapproach is not quantitative. In addition, because of large scatteramong replicate symmetric PCR amplifications, end-point assays ingeneral tend to be only qualitative.

Sequencing reaction products provides an alternative to probing.Traditional dideoxy sequencing may utilize products of amplificationreactions, such as symmetric PCR or LATE-PCR, as starting materials forcycle sequencing. Amplified product is purified utilizing ethanolprecipitation or an affinity column to remove leftover dNTPs andprimers, subjected to a cycle sequencing reaction utilizing onesequencing primer and fluorescently labeled dideoxy nucleotides, andsubjected to capillary gel electrophoresis. “Pyrosequencing” is areal-time, isothermal, sequence-by-synthesis method known in the art. Ifexponential amplification methods, for example PCR, are used in thepreparation of starting material for Pyrosequencing, amplified productmust be cleaned up by isolation of single-stranded product as well asremoval of dNTPs, pyrophosphate and unincorporated primers from theamplification reaction. LATE-PCR simplifies sample preparation, becauseit generates primarily single-stranded product, but it does not in andof itself eliminate the need to clean-up the product.

An aspect of this invention is methods for homogeneous detection ofreaction products of amplification reactions, temperature cycling orisothermal, utilizing the detection of fluorescence fromfluorophore-labeled linear oligonucleotide primers excited indirectly byexciting a DNA fluorescent dye such as SYBR Green I or, preferably, SYBRGold. Such dyes become fluorescent when they associate withdouble-stranded DNA, into which they are reported to intercalate. Theforegoing methods may be performed in real time or following theamplification reaction, either by reading fluorescence at a detectiontemperature (end-point detection) or by ascertaining changes influorescence as a function of temperature by post-amplification meltinganalysis. As a reaction mixture is heated through the meltingtemperatures of various reaction products, fluorescence decreasesprogressively as various amplicons containing a particularfluorophore-containing primer reach their melting temperatures andbecome single-stranded. Preferred methods include calculating the ratioof primer signal to dye signal.

Another aspect of this invention is reagent kits that include both DNAfluorescents dye and at least one such labeled primer, preferably aspart of a primer pair, and optionally amplification reagents.

Yet other aspects of this invention are homogeneous methods fordetecting reaction products of LATE-PCR reactions employing alow-temperature detection step. Certain embodiments comprise includingin the reaction mixture at least one allele-discriminating probeaccording to this invention, namely, a quenched double-stranded probegenerally of the type described by Li, Q. et al. (2002) Nucl. Acids Res.30: e5 except that it is a low temperature (Low-T_(m), or Super-LowT_(m)) target-specific probe and that it is excited indirectly byexciting a DNA fluorescent dye intercalated into the probe-target hybridsuch as, preferably, SYBR Gold. Other embodiments comprise including inthe reaction mixture at least one indirectly excitable mismatch-tolerantprobe according to this invention, namely, a quenched single-strandedprobe generally of the type described by Lee and Furst United Statespublished patent application Pub. No. US 2002/0119450 except that is aquenched low-temperature probe. These various methods include excitingthe dye during the low-temperature detection steps of a LATE-PCRamplification and detecting fluorescence from the probes under theseconditions to provide a measure of the target single-stranded sequence.Particular embodiments may further include measuring the total amount ofdouble-stranded product(s) in the reaction mixture by detecting dyefluorescence, preferably during or at the end of the extension step ofPCR cycles while the temperature of the reaction mixture is above themelting temperature(s) of the probes. Certain preferred methods includecalculating the ratio of probe signal to dye signal. In the case ofreplicate samples, such ratio corrects for differences among replicatesamples in amplification yields known to occur in PCR amplifications.

Other aspects of this invention are such low-temperatureallele-discriminating and quenched mismatch-tolerant probes, LATE-PCRkits that include at least one such low-temperature target-specificprobe together with amplification reagents and preferably thefluorescent DNA dye; and oligonucleotide sets comprising LATE-PCRprimers and at least one such probe.

Other aspects of this invention are homogeneous detection methods foruse when multiple amplicons are present or may be present, such methodcomprising including in a LATE-PCR amplification reaction mixture one ormore low-temperature mismatch-tolerant detection probes that, because oftheir low T_(m), do not interfere with amplification and are nothydrolysed (cut) by a DNA polymerase having 5′-3′ exonuclease activity,and that emit a fluorescent signal when hybridized and excited, eitherdirectly by a suitable excitation source or indirectly by a fluorescentDNA dye that is excited by a suitable excitation source. Such methodsinclude single-probe assays and multiple-probe assays for applicationssuch as genotyping. More than one probe may be labeled with the samefluorophore, in which event discrimination relies on change influorescence with temperature, just as when a single probe is used.Probes may be labeled with different fluorophores, in which event colordifference is also used for discrimination. Discrimination among targetsfor purposes of identification and quantification may includefluorescence ratios between fluorophores at the same or differenttemperatures, as well as fluorophore-to-dye ratios. Detection ispreferably performed during the amplification (real time) and morepreferably during a low-temperature detection step included in aLATE-PCR amplification protocol, and the detection step may includedetection at multiple temperatures. Yet another aspect of this inventionis a single-stranded linear probe useful in such detection methods, suchprobe being of the type described in U.S. patent application publicationU.S. 2002/0119450 (29 Aug. 2002), that is, a probe excited by thefluorescence emission from a fluorescent DNA dye, except that it is alow-temperature (Low-T_(m) or Super-Low-T_(m)) probe, ismismatch-tolerant, and includes a quenching moiety that quenches thefluorescence, which otherwise would result from secondary structure ofat low temperature.

Another aspect of this invention is an amplification-through-sequencingmethod that permits the product of a LATE-PCR amplification to beprepared for pyrosequencing in the amplification reaction chamber,vessel, well, slide or container, a “single-tube” operation, which maybe utilized with LATE-PCR amplifications performed in small volumes,preferably 17 ul or less.

Another aspect of this invention is a method for preparing LATE-PCRproducts for dideoxy sequencing utilizing only post-amplificationaqueous dilution of amplification reaction mixtures, which may beperformed as a “single-tube” operation.

SUMMARY

In this application references are made to melting temperatures, T_(m),of nucleic acid primers, probes and amplicons. T_(m), means thetemperature at which half of the subject material exists indouble-stranded form and the remainder is single stranded. Generally,except for LATE-PCR, the T_(m), of a primer is a calculated value usingeither the “% GC” method (Wetmar, J. G. (1991) “DNA Probes: Applicationsof the Principles of Nucleic Acid Hybridization,” Crit. Rev. Biochem.Mol. Biol. 26: 227-259) or the “2(A+T) plus 4(G+C)” method, both ofwhich are well known, at a standard condition of primer and saltconcentration. LATE-PCR, however, takes into account the actual primermelting temperatures in a particular reaction, taking into accountprimer concentrations at the start of amplification. See Sanchez et al.(2004) PNAS (USA) 101: 1933-1938, and Pierce et al. (2005) Proc. Natl.Acad. Sci (USA) 102: 8609-8614.

In this application we refer to such a concentration-adjusted meltingtemperature at the start of amplification as T_(m[0]), which can bedetermined empirically, as is necessary when non-natural nucleotides areused, or calculated according to the “nearest neighbor” method (SantaLucia, J. (1998), PNAS (USA) 95: 1460-1465; and Allawi, H. T. and SantaLucia, J. (1997), Biochem. 36: 10581-10594) using a salt concentrationadjustment, which in the examples below was 0.07 M monovalent cationconcentration. LATE-PCR may also take into account the meltingtemperature of the amplicon, which is calculated utilizing the formula:T_(m)=81.5+0.41(% G+% C)−500/L+16.6 log [M]/(1+0.7 [M]), where L is thelength in nucleotides and [M] is the molar concentration of monovalentcations. Melting temperatures of linear, or random-coil, probes arecalculated as for primers. Melting temperatures of structured probes,for example molecular beacon probes, can be determined empirically.

As used in this application, “LATE-PCR” means a non-symmetric DNAamplification employing the polymerase chain reaction (PCR) processutilizing one oligonucleotide primer (the “Excess Primer”) in at leastfive-fold excess with respect to the other primer (the “LimitingPrimer”), which itself is utilized at low concentration, up to 200 nM,so as to be exhausted in roughly sufficient PCR cycles to producefluorescently detectable double-stranded amplicon, wherein theconcentration-adjusted melting temperature of the Limiting Primer at thestart of amplification, T_(m[0]) ^(L), is not more than 5° C. below theconcentration-adjusted melting temperature of the Excess Primer at thestart of amplification, T_(m[0]) ^(X), preferably at least as high andmore preferably 3-10° C. higher; and wherein thermal cycling iscontinued for multiple cycles after exhaustion of the Limiting Primer toproduce single-stranded product, namely, the extension product of theExcess Primer, sometimes referred to as the “Excess Primer Strand”.

Primers and probes of this invention or useful in methods and kits ofthis invention are oligonucleotides in the broad sense, by which ismeant that they may be DNA, RNA, mixtures of DNA and RNA, and they mayinclude non-natural nucleotides (for example, 2′ o-methylribonucleotides) and non-natural internucleotide linkages (for example,phosphorothioate linkages). Both primers and probes function in part byhybridizing to a sequence of interest in a reaction mixture. A primer isa single-stranded oligonucleotide that can hybridize to itscomplementary sequence at the primer annealing temperature of anamplification reaction and be extended at its 3′ end by a DNApolymerase. A primer of this invention is a primer that signalshybridization of its priming sequence by means of a fluorophore that isindirectly excitable. A probe of this invention or useful in methods andkits of this invention is or includes a single-stranded oligonucleotidethat can hybridize to its intended target sequence (or sequences) at thedetection temperature (or temperatures) in or following an amplificationreaction and fluorescently signal that hybridization event by means of afluorophore that is indirectly excitable. As used herein a “probe” isnot extended in the amplification reaction by a DNA polymerase. Probesthat are very specific for a perfectly complementary target sequence andstrongly reject closely related sequences having one or a few mismatchedbases are “allele discriminating.” Probes that hybridize under at leastone applicable detection condition not only to perfectly complementarysequences but also to partially complementary sequences having one ormore mismatched bases are “mismatch tolerant” probes.

“Fluorescent DNA dye” as used herein means a composition, for exampleSYBR Green I or SYBR Gold, that becomes fluorescently excitable when itassociates with double-stranded DNA. It has been reported thatfluorescent DNA dyes intercalate into double-stranded DNA, but we do notwish to be bound by any theory of operation.

Primers of this invention are used in conjunction with a fluorescent DNAdye and are linear single-stranded oligonucleotides labeled with afluorophore that is indirectly excitable, that is, when the primerhybridizes to a template strand in the reaction mixture to form a regionof double-stranded DNA, and light (usually but not necessarily visiblelight) of a wavelength that excites, or is absorbed by, the DNAfluorescent dye but not the fluorophore is shone on the sample, thefluorophore emits. It has been reported that energy transfers from afluorescent DNA dye to a nearby fluorophore by fluorescence resonanceenergy transfer (FRET), but we do not wish to be bound by any theory ofoperation. We refer to a fluorophore that emits in this circumstance asa fluorophore that is “indirectly excited.” Probes of this invention arelikewise used in conjunction with a fluorescent dye that binds todouble-stranded DNA (a “fluorescent DNA dye”) and labeled with such anindirectly excitable fluorophore such that when the probe hybridizes toa target strand in the reaction mixture and the dye is excited, thefluorophore emits.

As used herein “kit” means a collection of reagents for performing anamplification or assay. A kit may be “complete”, that is, include allreagents needed for all steps of an amplification oramplification-detection. Alternatively a kit may be “partial”, omittingcertain reagents needed for those operations. Both complete and partialkits of this invention may additionally include reagents for samplepreparation, such as nucleic acid isolation and reverse transcription.Sequencing may involve two kits, for example, a complete LATE-PCRamplification kit and a complete cycle sequencing kit, or the two may becombined into a single kit.

As used herein an “oligonucleotide set” means a collection of primers orprimers and probes for performing an amplification or assay. Forsequencing an oligonucleotide set may include, for example, LimitingPrimer and Excess Primer for a LATE-PCR amplification and one or moreadditional sequencing primers for cycle sequencing. For a hybridizationprobe assay an oligonucleotide set may include, for example, LimitingPrimer and Excess Primer for a LATE-PCR amplification and at least onefluorophore-labeled hybridization probe.

As used herein a “single-tube” method means a series of at least twooperations, for example, sample preparation, amplification orsequencing, that can be performed without transferring the sample fromone container, be it a test tube, a reaction well, a chamber in amicrofluidics device, a glass slide, or any other apparatus capable ofholding a reaction mixture, to another container.

Probes that have low melting temperatures (that is, probes that formprobe-target hybrids having low melting temperatures) can be added toamplification reaction mixtures prior to the start of amplification andutilized only when desired. By keeping temperatures above the meltingtemperature of a probe during all or portions of an amplificationreaction, the probe is kept from hybridizing to its target and possiblyreducing the efficiency of the reaction. Certain embodiments of LATE-PCRassays utilize low temperature probes. As used herein, “Low-T_(m),probe” means a hybridization probe that has a concentration-adjustedmelting temperature at the start of amplification, T_(m[0]), at least 5°C. below the T_(m[0]) of the Limiting Primer in a LATE-PCR assay; and a“Super-Low-T_(m), probe” means a hybridization probe that has a T_(m[0])that is at least 5° C. below the mean primer annealing temperature ofthe exponential phase of a LATE-PCR reaction. We frequently add probesto LATE-PCR reactions at 1 micromolar (μM) concentration. Therefore,when designing probes, we sometimes utilize a nominal T_(m[0])calculated as described earlier but utilizing a nominal concentration of1 μM. Most Low-T_(m) and Super-Low-T_(m) probes have a T_(m[0])calculated at 1 TM concentration in the range of 30-55° C.

Detection utilizing low temperature probes requires low temperaturedetection, wherein the temperature of the probe-target mixture islowered sufficiently for fluorescently labeled probes to hybridize andsignal. This can be done at the conclusion of amplification (end point)or in a post-amplification melting analysis. Alternatively alow-temperature detection step may be included in some or all cycles ofthe linear phase of a LATE-PCR amplification for a real-time assay.Preferably such a step occurs after primer extension and beforehigh-temperature strand melting (or “denaturation”), although it couldbe included in the primer annealing step. A low-temperature detectionstep in a LATE-PCR assay signifies a reduction in temperature at least5° C. below the primer annealing temperature.

Certain methods according to this invention utilize fluorophore-labeledprimers or hybridization probes in combination with fluorescent dyesthat bind to double-stranded DNA and include stimulating a dye at awavelength that excites the dye but not the fluorophore(s) and detectingfluorescence emitted by a fluorophore stimulated indirectly by thisprocedure. Some embodiments of methods according to this inventioninclude detecting fluorescence emission from the dye as well. Certainpreferred methods further include calculating the ratio of fluorophoreemission to dye emission.

One embodiment of this invention includes adding to a nucleic acidamplification mixture a fluorescent DNA dye, such as SYBR Green I, orpreferably SYBR Gold, and at least one amplification primer according tothis invention, that is, a linear single-stranded oligonucleotide thatis extendable by a DNA polymerase and that is labeled with a fluorophorethat is indirectly excitable to signal priming as described above;performing an amplification reaction, preferably a PCR reaction(including LATE-PCR), that includes annealing and extending that labeledprimer; and either during the amplification (real-time detection) orfollowing completion of amplification (either an end-point detection atthe conclusion of the amplification reaction or during a subsequentthermal analysis (melting curve)) exciting the dye and detectingfluorescence emission from the fluorophore, either alone or incombination with detecting fluorescence emission from the dye. Byappropriate amplification protocol design, melting analysis ofdouble-stranded products can be included at desired points in anamplification reaction. In this embodiment only primers that areincorporated into double-stranded DNA will fluoresce. Unincorporatedprimers will not fluoresce, so there is no need to separate unboundprimers physically. The method is homogeneous. Also, fluorophoreemission comes only from double-stranded regions of products thatinclude a labeled primer, not from all double-stranded products. Example1 below demonstrates these improvements. It shows that in asingle-extension cycle designed to produce mixed extension products ofvarious lengths, a melting curve based on detection of emissions fromthe primer's fluorophore showed all products, whereas a melting curvebased on detection of emissions from the dye did not. Example 1demonstrates also the use of the method of this embodiment in isothermalreactions.

As will be appreciated by a person versed in the art, it is generallyimportant to correct for fluorescence overlap when a fluorescent DNAdye, for example SYBR Green I, is used in conjunction with afluorescently labeled primer or probe that is excited by FRET from theintercalated dye. This is the case because fluorescent DNA dyestypically emit light over a broad spectral range which may include thewavelength of light used to measure the fluorescence emitted by theprimer or probe. The desired correction can be achieved by: 1)establishing the emission spectrum of the dye alone; 2) measuring theintensity of the dye emission in each sample at a wavelength that isshorter than the emission wavelength of the primer or probe; 3)calculating the intensity of the dye emission at the emission wavelengthof the primer or probe on the knowledge of steps 1 and 2; and 4)subtracting that calculated dye intensity from the total intensitymeasured at the emission wavelength of the primer or probe. Manycommercially machines, such as the ABI 7700 and the Cepheid Smart Cyclerprovide software for carrying out this correction. Alternatively themeasurements of dye spectrum, dye emission alone, and total dye/probeemission can be made and a satisfactory formula for correction can bemanually applied. For instance, Lee and Fuerst, United States PublishedPatent Application Pub. No. US 2002/0119450 describes such a formula formeasurement and manual correction of SYBR Green I fluorescence overlapon the Light Cycler.

All of the Examples described in this application were carried out onthe ABI 7700 and machine software was used to correct for fluorescenceoverlap in all cases in which a fluorescent DNA dye was used inconjunction with an indirectly excited fluorescent primer or probe,regardless of whether the fluorescence of the dye alone was recorded.

For PCR amplifications utilizing a single primer pair, wherein at leastone primer is fluorophore-labeled for indirect excitation as describedabove, a melt-curve analysis according to this embodiment candistinguish between the intended product(s) and non-specific products.For multiplex PCR amplifications utilizing multiple primer pairs,wherein at least one member of each pair is fluorophore-labeled and adifferent fluorophore is utilized for each pair, different intendedproducts can be distinguished by color and by the melting temperaturesassociated with the different fluorophores. For PCR amplificationsgenerally, fluorophore emission(s) and dye emissions can be monitoredduring the reaction to track the build-up of specific products(s) and totrack the build-up of all double-stranded products, respectively.

Analyses of amplification reactions may utilize the ratio of fluorophoreemissions, a signal specific to hybridized primers or probes, to thedye-emission signal, which is not so specific. Such a ratio, forexample, corrects for variations among replicate reactions. Also,analyses may utilize the primer-template melting peak, which decreasesin magnitude as labeled primer is incorporated into extension product orproducts.

This invention includes amplification kits and partial kits that includea fluorescent DNA dye, at least one primer pair that includes a primerlabeled with a fluorophore that is excited indirectly when the dye isexcited, and reagents to amplify the region defined by the primers,preferably by LATE-PCR.

Another embodiment of a method according to this invention includesadding to a nucleic acid amplification mixture a fluorescent DNA dye,such as SYBR Green I or, preferably, SYBR Gold, and at least oneindirectly excitable, quenched, allele-discriminating Low-T_(m) orSuper-Low-T_(m) hybridization probe, which may be a probe of thisinvention. Allele-discriminating probes of this invention are the typeof double-stranded probes described by Li, Q. et al. (2002), “A NewClass of Homogeneous Nucleic Acid Probes Based on Specific DisplacementHybridization,” Nucl. Acid Res. 30: (2)e5 (a fluorophore-labeled linearoligonucleotide probe strand complementary to the target, and aquencher-labeled complementary strand that is shorter than the probestrand, generally by 2-10 nucleotides), except that they are labeledwith a fluorophore that is excited indirectly by exciting the dye, andthat they have a low melting temperature suitable for use in LATE-PCRamplifications as Low-T_(m) or Super-Low-T_(m) probes.Allele-discriminating capacity of double-stranded probes can be adjustedas has been described by Li et al., as can the level of backgroundfluorescence. In addition, background fluorescence can be reduced byincluding guanidine residues adjacent to the fluorescent moiety,so-called “G-quenching.”

Methods of this embodiment include amplification utilizing such amixture and detection at a temperature at which the probe hybridizes inan allele-discriminating fashion. Preferred embodiments include using alow-temperature detection step during the linear amplification phase ofa LATE-PCR reaction wherein the foregoing probes hybridize to thesingle-stranded amplicon being synthesized, exciting the fluorescent DNAdye at a wavelength that does not excite the fluorophore or fluorophoresdirectly, and reading fluorescence from the probe's fluorophore orprobes' fluorophores, which is or are excited indirectly in thisfashion. Other embodiments include amplification followed by alow-temperature detection as an end-point determination. Someembodiments further include detecting emission from the dye, and certainpreferred embodiments include calculating a ratio of probe(s) emissionto dye emission. Detection of dye emission is most preferably performedat the very start of the detection step, while the temperature of thereaction mixture is above the melting temperatures of all probes thatare present. Data from accumulating or accumulated double-strandedmolecules (the dye signal) and from accumulating or accumulatedsingle-stranded molecules (the signal from each probe) can be used toconstruct ratios in the manner described. Methods of this embodimentalso include use of low-temperature molecular beacon probes, asdescribed in published application WO 03/054233, if the fluorophorelabel is stimulated by emission from the dye but not by the wavelengthused to excite the dye.

This invention also includes LATE-PCR assay kits and partial kits thatinclude reagents for performing a non-symmetric amplification,preferably a LATE-PCR amplification, with a low temperature detectionstep (end point or real time) and that include a fluorescent DNA dye, atleast one primer pair, preferably a LATE-PCR primer pair including anExcess Primer and Limiting Primer, and at least one fluorophore-labeledLow-T_(m) or Super-Low-T_(m) hybridization probe for a single-strandedproduct of the amplification reaction (extension product of the primerpresent in excess), wherein the probe is not mismatch tolerant butrather is allele-discriminating at the intended detection temperature,and wherein the probe's fluorophore is indirectly excited by excitationof the dye. In preferred kits and partial kits, at least one probe is anallele-discriminating probe of this invention. This invention alsoincludes oligonucleotide sets that include at least one pair of primersfor non-symmetric amplification, preferably LATE-PCR amplification, andat least one Low-T_(m) or Super-Low-T_(m) quenched allele-discriminatingdouble-stranded probe labeled with a fluorophore so as to be indirectlyexcitable as described above, preferably by a SYBR dye, as well as suchdouble-stranded probes themselves.

Yet another embodiment of a method according to this invention includesadding to a non-symmetric amplification reaction mixture, preferably aLATE-PCR reaction mixture, detection reagents comprising a fluorescentDNA dye such as SYBR Gold and at least one mismatch-tolerantsingle-stranded linear hybridization probe that is perfectlycomplementary to one possible single-stranded amplicon target sequencethat may or may not be present for amplification in the reaction and isless than perfectly complementary to at least one other possiblesingle-stranded amplicon target sequence that may be present. Probesuseful in this embodiment are single strands labeled with a fluorophorethat is indirectly excitable by fluorescence emission from the dye. Theyare Low-T_(m) or, preferably, Super-Low-T_(m) probes with respect totheir most complementary possible targets that may be present, generallymeaning perfectly matched target. It is preferred that they have aT_(m[0]) against perfectly complementary target that is not more than afew degrees higher, and preferably below, more preferably at least 5° C.below, the primer annealing temperature during the exponentialamplification phase of the amplification reaction. The probes may belinear (or random-coil) probes, or random-coil probes according to thisinvention, that is, quenched to eliminate signal due to formation ofsecondary structure at low temperatures. Quenched linear probesaccording to this invention preferably have a fluorophore on one end anda non-fluorescent quenching moiety on the other end, the one on the 3′end of the probe replacing the phosphate cap otherwise added to preventthe probe from being extended, that is, functioning as a primer.

This embodiment comprises subjecting the foregoing mixture tonon-symmetric, preferably LATE-PCR, amplification to generatesingle-stranded amplicon molecules and subjecting the amplificationreaction mixture to a thermal analysis utilizing at least one mismatchtolerant probe that signals upon hybridization. Thermal analysis can beperformed not only after the amplification reaction is completed butalso during a LATE-PCR low-temperature detection step during thermalcycles in which single-stranded product is being produced, that is,after exhaustion of the Limiting Primer. Thermal analysis revealstargets of each probe according to the melting temperatures of theprobe-target hybrids that form or destabilize as the temperature islowered or raised, respectively. As the temperature is lowered, a probewill first hybridize to its perfectly matched target (if present) andemit a fluorescent signal. As the temperature is lowered further, theprobe will hybridize successively to progressively “more mismatched”targets and emit increased fluorescent signal on each occasion. Asexplained in connection with previous embodiments, emission from thefluorescent DNA dye can also be detected, preferably when probes are nothybridized, that is, at a temperature above the T_(m) of the probe(s),to permit monitoring of the accumulation of double-stranded molecules inthe reaction and to permit the use of ratios to reduce scatter amongreplicate samples.

This invention includes kits containing reagents for non-symmetricamplification, preferably a LATE-PCR amplification, that include afluorescent DNA dye, at least one primer pair, preferably a LATE-PCRprimer pair including an Excess Primer and a Limiting Primer, and atleast one mismatch-tolerant Low-T_(m) or Super-Low-T_(m) random coilprobe, quenched if necessary, for a single-stranded amplificationproduct(s), as well as partial kits and oligonucleotide sets containingsuch primers and probes, and such probes themselves.

Methods according to this invention that utilize a low-temperaturedetection step of LATE-PCR assays, preferably a low-temperaturedetection step following primer extension and before strand melting,include multiplex probe assays which contain more than one pair ofprimers and generate one or more single-stranded amplicons (one probefor each target) as well as multiprobe assays that contain at least oneprobe for multiple targets. Certain preferred methods with alow-temperature detection step include a low-temperature detection stepfollowing primer extension and before strand melting. During thedetection step in such assays the temperature may be dropped as much as30° C. or even 40° C. below the primer annealing temperature, providinga large temperature window for detection. Allele-discriminating probes,in addition to being differentiable by color (fluorophore emissionwavelength) can be differentiated by differences in melting temperature.For example, four different FAM-labeled allele-discriminating probeswith T_(m)'s of 30, 35, 40 and 45° C., respectively, against theirtargets can be distinguished in real time or following amplification asan end-point determination, as the reaction temperature is lowered orraised, not just by post-amplification melt analysis. This added degreeof freedom multiplies significantly the number of different probes thatcan be used in the same reaction. Mismatch-tolerant probes will havelower T_(m)'s against mismatched targets than against perfectly matchedtargets. Combinations of differently colored low-temperaturemismatch-tolerant probes that signal upon hybridization produce patternsof temperature-dependent fluorescence emission curves duringlow-temperature detection. Methods according to this invention includeuse of such emission curves, derivative curves, and ratios of either ofthem at one temperature or different temperatures to identify theconstituents of mixed targets with post amplification melt analysis andalso in real time by monitoring fluorescence at several temperatureswithin the window of LATE-PCR low-temperature detection step. Ratios mayinclude same probe/probe, different probe/probe ratios, probe/dyeratios, and combinations thereof.

LATE-PCR kits, partial kits and oligonucleotide sets may include atleast two allele-discriminating probes of the same color that can bedistinguished by T_(m) or at least two mismatch-tolerant probes whosehybridization to different targets can be distinguished by T_(m),preferably quenched random-coil probes that are indirectly excited byexciting a fluorescent DNA dye.

This invention includes improved methods for preparing the amplificationproducts of LATE-PCR amplifications for sequencing reactions, eitherdideoxy sequencing or sequencing-by-synthesis methods such aspyrosequencing. In particular, we have demonstrated the generation andpreparation of such starting materials in a single reaction container,for example, a microcentrifuge tube. Preferred embodiments include inthe LATE-PCR reaction mixture a reagent for inhibiting mispriming, mostpreferably a reagent disclosed in our U.S. Provisional patentapplication No. 60/619,620, titled “Reagents and Methods for ImprovingReproducibility and Reducing Mispriming in PCR Amplification,” which isincorporated by reference herein in its entirety. For dideoxy sequencingwe have demonstrated preparing LATE-PCR amplification products forsequencing by the single step of sample dilution, a method we refer toas “dilute and go.” For pyrosequencing, we have demonstrated methodsthat require only addition of pyrosequencing enzyme/substrate reagentsto the LATE-PCR product mixture prior to primer annealing. In someembodiments, the amplification product of a LATE PCR process is cleanedup by diluting the amplification product by a factor of at least five(e.g., at least 8-fold).

Methods according to this invention also include LATE-PCR amplificationand sample preparation for Pyrosequencing in the same container, such asthe same reaction tube or the same chamber of a microfluidics device,all of which we refer to for short as “single-tube” methods. Intraditional Pyrosequencing, DNA is amplified by symmetric PCR where oneprimer is 5′ labeled with a biotin molecule. After amplification,streptavidin coated beads are used in conjunction with vacuum ormagnetic equipment to isolate single-stranded DNA (ssDNA) and wash awayresidual components of the PCR reaction that interfere withPyrosequencing including pyrophosphate (PPi), dNTPs and PCR primers. Byvirtue of its ability to generate ssDNA, LATE-PCR eliminates the needfor strand separation and simplifies sample preparation when combinedwith a same-container method for eliminating the four interferingcomponents left over from PCR. In one such method, the need to removedNTPs remaining at the end of amplification is minimized by usinglimiting amounts of dNTPs in the LATE-PCR amplification reactionmixture, care being taken to utilize a sufficient amount to produceenough ssDNA for Pyrosequencing. An enzyme with pyrophosphataseactivity, for example a pyrophosphatase such as yeast pyrophosphatase,is added to the amplification product to remove PPi and the mixture isheated to denature that enzyme before proceeding to Pyrosequencing.Because Limiting Primer does not remain after LATE-PCR amplification andthe residual Excess Primer cannot prime the strand extended from theExcess Primer during amplification (Excess Primer strand), leftoverprimers need not be removed in many cases. However, potential misprimingcan be avoided by including in the LATE-PCR reaction mixture anoligonucleotide that hybridizes to the Excess Primer at temperaturesbelow the T_(m) of the Excess Primer, including the temperature used forPyrosequencing. Alternatively, an oligonucleotide blocked for extensionat the 3′ end and fully complementary to the Excess Primer can be addedafter LATE-PCR amplification but before Pyrosequencing to avoidpotential mispriming by the Excess Primer at temperatures used forPyrosequencing. A third strategy to avoid mispriming by the ExcessPrimer at the 3′ end of the strand extended from the Limiting Primerduring amplification (Limiting Primer strand) involves using asufficient concentration of a 3′ blocked oligonucleotide containing thesame sequence as the Excess Primer to out-compete the Excess Primer forbinding sites.

Our more preferred method of “single-tube” sample preparation avoids theneed to determine appropriate limiting dNTP concentrations forparticular amplifications. In this method we first add Pyrosequencingenzyme/substrate reagents to the LATE-PCR product, which removes dNTPsand PPi. We follow this with primer annealing using an added sequencingprimer and then add individual dNTPs for Pyrosequencing. Alternatively,one may eliminate dNTPs by addition of a purified enzyme with a dNTPaseactivity, such as potato apyrase, followed by heating to inactivate theenzyme and one may eliminate pyrophosphate by addition of a purifiedenzyme with pyrophosphatase activity, such as yeast pyrophosphatase,followed by heating to inactivate the enzyme. If both enzymes areemployed they can be added at the same time.

Assays according to this invention particularly LATE-PCR assays,preferably include means to avoid mispriming, which can cause a decreasein probe signal in the late stages of the reaction. We have successfullyavoided this “hook effect” by including in the reaction mixture amispriming-suppressing reagent disclosed in our United StatesProvisional patent application described above. We have also avoidedthat effect by adjusting the concentration of polymerase added to thereaction. Decreasing mispriming by adjusting polymerase can be observedin terms of the kinetics of the LATE-PCR reaction using a probe of thessDNA, as well as by the composition of the final product revealed byvarious means known in the art.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B show the use of fluorescently labeled primers accordingto the methods of the invention for melting curve analysis.

FIGS. 2A and 2B show reduction of signal scatter through the use ofratios of single-stranded product to double-stranded product accordingto the methods of the invention.

FIGS. 3A, 3B, and 3C show comparison of identification of five speciesof Mycobacteria via melting curve analysis obtained with eitherconventional mismatch-tolerant probes against the 16S ribosomal RNA geneor two different versions of quenched mismatch-tolerant probes againstthe same target designed according to the methods of the invention.

FIGS. 4A, 4B, and 4C show shows identification of five species ofMycobacteria using only two mismatch-tolerant probes against the 16Sribosomal RNA gene according to the methods of the invention.

FIGS. 5A and 5B shows identification of five species of Mycobacteria viafirst derivative analysis of melting curves shown in using twomismatch-tolerant probes against the 16S ribosomal RNA gene designedaccording to FIGS. 3A and 3B the methods of the invention.

FIG. 6 shows identification of five species of Mycobacteria using ratiosof fluorescent signals collected at different temperatures from twomismatch-tolerant probes against the 16S ribosomal RNA gene according tothe methods of the invention.

FIG. 7 shows end-point genotyping of homozygous and heterozygous samplesfor the G269 mutation of the human HexA gene using LATE-PCR and a singleLow-T_(m) mismatch-tolerant probe against the wild-type allele accordingto the methods of the invention.

FIG. 8 shows separate identification of three different alleles of thehuman cystic fibrosis transmembrane regulator (CFTR) gene usingLATE-PCR, allele discriminating Low-T_(m) probes labeled with the samecolor, and first-derivative analysis of melting curves.

FIG. 9 shows simultaneous identification of different combinations ofvarious alleles of the human cystic fibrosis transmembrane regulator(CFTR) gene using allele discriminating Low-T_(m) probes labeled withthe same color, and first-derivative analysis of melting curves.

FIG. 10 shows identification of different allele combinations of thehuman cystic fibrosis transmembrane regulator (CFTR) gene by plottingthe changes in fluorescence at two temperatures collected according tothe methods of the invention.

FIG. 11 shows Two Temperature Normalization assays (with backgroundcorrection)

FIG. 12 shows Two Temperature Normalization assays (without backgroundcorrection)

FIGS. 13A, 13B, and 13C show Three Temperature Normalization assays

FIG. 14 shows pyrograms of the “dilute-and-go” method of preparation ofLATE-PCR samples for pyrosequencing according the methods of theinvention (A) relative to the conventional method of preparation ofLATE-PCR samples (B) for the same assay, and (C) a comparison thereof.

FIG. 15 is Pyrograms obtained from single cells prepared by thesingle-tube LATE-PCR method. Arrows indicate the β-globin IVS 110 siteof: (A) homozygous wild-type, (B) heterozygous and (C) homozygous mutantcells.

FIG. 16 is the Pyrogram from a Pyrosequencing reaction carried out formore than fifty base pairs. Nucleotide dispensation order is listedbelow each peak and the expected sequence is noted above.

FIG. 17 is dideoxy sequencing chromatographs resulting from the“dilute-and-go” method of preparation of LATE-PCR samples for dideoxysequencing according the methods of the invention and from theconventional method of preparation of LATE-PCR samples for the sameassay for (A) SEQ ID NO: 43, (B) SEQ ID NO: 44, (C) SEQ ID NO: 45, and(D) SEQ ID NO: 46.

FIG. 18 is an electrophoresis gel from a LATE-PCR amplification of morethan one product from the same DNA template in the same reaction.

FIG. 19 is chromatographs ((A) SEQ ID NO: 47, (B) SEQ ID NO: 48, (C) SEQID NO: 49, and (D) SEQ ID NO: 50) from dilute-and-go dideoxy sequencingof the product of the LATE-PCR amplification of FIG. 18.

FIG. 20 shows that (A) the amount of ssDNA and dsDNA generated by aLATE-PCR amplification can be measured independently and (B) can be usedto calculate the ratio ssDNA/dsDNA which, in turn, can be used todetermine whether the amount of ssDNA thus far accumulated is sufficientfor subsequent sequencing via the “dilute-and-go” method.

FIG. 21 is dideoxy sequencing chromatographs resulting from the“dilute-and-go” method employed on a 50:50 mixture of LATE-PCR ampliconshaving two closely related, but different sequences.

FIG. 22 shows the sensitivity range of mixed LATE-PCR amplicons havingclosely related, but different sequences that can be distinguished viathe “dilute-and-go” method.

FIG. 23 shows that a LATE-PCR together with at least one singlemismatch-tolerant probe can be used to (A) generate end-point meltingcurves which, in turn, can be used to (B) quantify the relative amountsof two or more mixed LATE-PCR amplicons having closely related, butdifferent sequences.

FIG. 24 shows the kinetics of several LATE-PCR assays carried out usingtwo different concentrations of Taq polymerase with each of threedifferent amounts of genomic DNA.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

This invention includes nucleic acid amplification assays, for examplePCR assays, that include detection of fluorescence emission from atleast one fluorophore-labeled primer that is excited, not directly byapplying light (visible or not) of a wavelength strongly absorbed by thefluorophore, but indirectly by applying light of a wavelength thatexcites a nearby fluorescent DNA dye such as SYBR Green or, preferably,SYBR Gold, as well as complete and partial kits containing all or someamplification reagents and oligonucleotide sets containing such labeledprimers, and also the primers themselves.

Amplification primers are well known. Primers according to thisinvention are short oligonucleotides, generally under fifty bases inlength that hybridize to a target strand and are extended by anappropriate polymerase. A primer may be composed of naturally occurringnucleotides, or it may include non-natural nucleotides and non-naturalinternucleotide linkages. Although primers are generally linearoligonucleotides, they may include secondary structure. (See, forexample, Nazarenko I A, Bhatnagar S K, Hohman R J (1997), “A Closed TubeFormat for Amplification and Detection of DNA Based on Energy Transfer,”Nucleic Acids Res. 25:2516-2521). Amplifications often include use ofone or more primer pairs each consisting of a forward primer and areverse primer. In methods, kits and oligonucleotide sets according tothis invention, either one primer of a pair or both primers of the pairmay be labeled with a covalently bound fluorophore that fluoresces whennearby fluorescent DNA dye is stimulated. When the labeled primerhybridizes (or anneals) to its complementary sequence in a templatestrand, a double-stranded region is formed. Fluorescent DNA dyeassociates with that region, by intercalating therein or otherwise, andbecomes fluorescent in that region, which is nearby to the primer'sfluorophore such that when the dye is stimulated at a wavelength thatdoes not directly excite the fluorophore, the fluorophore emits at itscharacteristic wavelength. These primers may be used to monitorsynthesis of products resulting by extension of a DNA polymerase such asthose resulting from PCR and primer extension assays in real-time or byend-point detection and/or to assess product specificity by meltingcurve analysis.

Primers according to this invention, used as a substrate for extensionby a DNA polymerase, including primers for PCR amplification (symmetricor non-symmetric, including particularly LATE-PCR), are labeled at anynucleotide position with a covalently bound fluorophore such that the 3′end of the oligonucleotide primer remains available for extension. Theprimers can have the design of double-stranded probes described by Li,Q. et al. (2002) (“A New Class of Homogeneous Nucleic Acid Probes Basedon Specific Displacement Hybridization,” Nucl. Acid Res. 30: (2)e5). Theonly sequence constraint on the oligonucleotide of the primer is thatthe oligonucleotide must not have any secondary structure that itselfleads to indirect fluorophore excitation, meaning that generally thereis not secondary structure greater than 2 base pairs. The fluorophoremoiety should not be appreciably excited directly by, but the dye mustbe directly excited by, the excitation source wavelength used; thefluorophore must emit when the fluorescent DNA dye is excited in itsimmediate presence, generally not greater than a distance at which thefluorophore undergoes fluorescence resonance energy transfer (FRET)occurs; and the emission spectrum of the chosen fluorophore must bedistinguishable from the emission spectrum of the fluorescent DNA dyeeither by the use of filters or spectral deconvolution. Under theseconditions, the fluorophore fluoresces upon incorporation into doublestranded product following primer annealing, including extension by aDNA polymerase. Loss of fluorescence takes place during heating when atthe melting temperature (T_(m)) of the particular stretch ofdouble-stranded DNA containing the fluorophore is reached.

Conditions for the use of primers according to this invention inconjunction with fluorescent DNA dyes (primer and DNA dye concentration,DNA dye excitation wavelength) are the same as those known in the artfor monitoring the synthesis of products of primer extension reactions(including PCR) in the course of the reaction and for assessingextension product specificity by melting curve analysis using onlyfluorescent DNA dyes with the exception that fluorescence is collectedat the emission wavelength corresponding to the primer fluorophoreinstead of or in addition to the emission wavelength of the dye. Underthese conditions, the fluorescence signals originate fromdouble-stranded sequences containing the primers, rather than alldouble-stranded sequences in the reaction.

Comparison of the performance of DNA dye to methods and systemsaccording to this invention was performed by the experiment reportedbelow in Example 1 and in FIG. 1. A fluorophore-labeled primer wasextended by DNA polymerase in the presence of SYBR Green dye and in thepresence of a relatively long non-extendable oligonucleotide hybridizedto the template strand near to the region of primer extension. Thisresulted in a product mixture having template strand-unextended primerhybrids, short primer-extension products, and the non-extendableoligonucleotide, such that hybrids with the template had T_(m)'s rangingfrom 60° C. (the fluorophore (Cy5)-labeled primer) to 79° C. (thenon-extendable oligonucleotide), with primer-extension products fallingbetween those two T_(m)'s.

Standard melt-curve analysis was performed on the final reaction mixture(duplicate samples) using both fluorescence readings from the dye andfluorescence readings from the fluorophore. Melting curves are presentedin FIG. 1. Panel A is the melt curves 1 obtained utilizing dyeemissions. The sole peak is 79° C., the melting temperature of thenonextendable oligonucleotide. No other peak is seen, not even that ofthe unextended primer. Panel A demonstrates the migration of SYBR Greendye to the higher T_(m) hybrid during generation of a melt curve, whichmasks the presence of lower T_(m) hybrids. Panel B is the melt curves 2obtained utilizing fluorophore emissions. It shows a peak at 60° C., theT_(m) of unextended primer-template hybrid, and an additional peak at atemperature between 69° C. and 79° C., that is, a peak indicative ofprimer extension product. The lower T_(m)'s are seen despite thetendency of the dye to migrate, as shown by melt curves 1. Monitoringfluorophore emission according to this invention reveals every hybridspecies labeled with the fluorophore in the mixture at its correctconcentration.

In the case of PCR amplifications utilizing a single pair of primers,wherein at least one member of the pair is a primer according to thisinvention, melt curve analysis can distinguish between specific andnon-specific products using a single fluor because the specific producthas an expected melting temperature and the non-specific product has anunexpected, melting temperature. In the case of multiplex PCRamplifications, utilizing more than one pair of primers, wherein atleast one member of each pair of primers is a primer according to thisinvention, two different specific products can be distinguished fromeach other either because they have different, but expected, T_(m)values and or because the two different primers employed are labeledwith different fluorophores. Moreover, melting curve analysis usingprimers according to this invention can be carried out during an ongoingamplification reaction or at the end of a reaction.

Incorporation of one or more primers according to this invention duringthe course of a reaction can also be used to measure quantitatively theextent of amplification of one or more targets during the course of aPCR, or the synthesis of one or more stretches of double-stranded DNAduring the course of an isothermal extension reaction. In either case,the amount of the full-length double-stranded product molecule ormolecules can be followed over time by repeated detection of increasingfluorescence, or can be measured at the end of a reaction. In addition,incorporation of one or more primers according to this invention duringthe course of either isothermal reactions or thermal cycled reactionscan be used to measure existence and/or accumulation of partialproducts, i.e. those that have begun extension along a template strandbut have not reached their maximum possible length. In such cases themelting temperatures of the partial products are lower than the meltingtemperature of the full-length product, but are higher than the meltingtemperature of the labeled primer from which they are derived. Inaddition, concomitant with incorporation of the labeled primer into apartial or full-length product strand, the magnitude of the meltingtemperature peak generated from the primer/template DNA-DNA hybriddecreases, and can be used as an additional measure of DNA synthesis.

As stated above, each stretch of double-stranded DNA or ampliconsynthesized by incorporation of a primer according to this inventiongenerates a fluorescent signal at the emission wavelength of thecovalently bound fluorophore of the primer, when indirectly stimulatedby FRET or other mechanism from the bound SYBR dye, a“primer-specific-signal”. The same double-stranded DNA also generates afluorescent signal at the emission wavelength of the SYBR dye, the“total-SYBR-signal”, the sum of all double-stranded sequences present inthe reaction, since all double-stranded sequences fluoresce, regardlessof whether they have an incorporated labeled primer. Thus, primersaccording to this invention can be used to analyze the fluorescentsignals in terms of the following ratio:(primer-specific-signal/total-SYBR-signal), hereafter the (PSS/TSS)value. Data analysis in terms of the (PSS/TSS) value corrects forvariations in fluorescent DNA dye signal (TSS) among replicatereactions. This is particularly useful in the case of LATE-PCRamplifications because the rate of single-stranded amplicon synthesis isproportional to the amount of double-stranded amplicon accumulated atthe end of the exponential phase of the reaction. Thus, smalldifferences in the level of double-stranded DNA among replicatereactions alter the rate of single-stranded amplicon accumulation.

It is also possible to utilize more than one primer labeled with thesame fluorophore, as long as the amplicons are differentiable by apost-amplification melting-curve analysis. See FIG. 1, Panel B, forexemplification of this principle. Signal from the common fluorophore atthe end of an extension step, which may be the final extension step (endpoint) or intermediate extension steps, gives an indication of totalamplicons incorporating the fluorophore. Melt-curve analysisdistinguishes among products and provides a quantitative measure oftheir concentrations.

LATE-PCR is a non-symmetric PCR amplification that, among otheradvantages, provides a large “temperature space” in which actions may betaken. See WO 03/054233 and Sanchez et al. (2004), cited above. LATE-PCRpermits the use of “Low-T_(m)” and “Super-Low T_(m)” hybridizationprobes to detect amplification products (“amplicons”) that aresingle-stranded. Various types of probes that are single-target-specificin a particular assay, including allele-discriminating probes capable ofdiscriminating against a single base-pair mismatch, such asallele-discriminating molecular beacon probes, can be utilized withLATE-PCR as Low-T_(m) and Super-Low T_(m) probes, as canmismatch-tolerant probes such as mismatch-tolerant molecular beaconprobes or linear (random-coil) probes having a fluorophore excitableindirectly by emission from a SYBR dye. We have devised a new class ofallele-discriminating probes useful as Low-T_(m) and Super-Low T_(m)probes in LATE-PCR assays that permit the determination ofsingle-stranded/double-stranded ratios within a reaction, as canallele-discriminating molecular beacon probes labeled with such afluorophore.

Allele-discriminating probes according to this invention are modifieddouble-stranded, allele-discriminating, quenched probes according to Li,Q. et al. (2002), Nucl. Acid Res. 30: (2)e5). They have the followingmodifications: they are labeled with a fluorophore that is indirectlyexcitable by exciting a double-stranded DNA fluorescent dye such as SYBRGreen or SBYR Gold but not directly excitable by wavelength utilized tostimulate the dye (in this regard similar to the primers discussedabove), and they are constructed to be Low-T_(m) or Super-Low T_(m)probes. When not bound to its target sequence, such a probe binds to ashorter complementary oligonucleotide. We prefer that the complementaryoligonucleotide include a quencher such as Dabcyl or a Black Hole™quencher to reduce background fluorescence from the probe. Alternativelyor in addition, background fluorescence can be reduced by includingguanidine residues adjacent to the fluorophore (G-quenching). In thepresence of fully complementary target strand, the shorter complementarystrand is displaced, the longer fluorophore-labeled strand hybridizes tothe target, and the fluorophore is unquenched and rendered capable ofreceiving energy from the dye so as to fluoresce at its characteristicwavelength. Several of these probes for different targets, labeled withdifferent fluorophores, can be used for multiplex assays.

Such allele-discriminating probes are designed to have aconcentration-adjusted melting temperature, T_(m[0]), in the assay thatmakes it a Low-T_(m) or Super-Low T_(m). The T_(m[0]) of theprobe-target hybrid is conveniently determined and adjusted empirically,although a calculated value may be employed at least as a good startingpoint to minimize adjustment. The length and concentration of thecomplementary probe strand relative to the fluorophore-labeled strandare adjusted empirically for maximal allele discrimination. We startwith a length 1-3 nucleotides shorter than the fluorophore-labeledstrand and a concentration of 1-1.2 times the concentration of thefluorophore-labeled strand.

In a LATE-PCR assay, these allele-discriminating probes are utilized ina low-temperature detection step, preferably following the primerextension step in cycles following exhaustion of the Limiting Primer.For real-time readings over multiple cycles, the SYBR dye is excited andfluorescence is read both from both the dye and from the fluorophore (orfluorophores). We prefer to read the dye signal during or at theconclusion of the PCR extension step when the temperature is above theT_(m) of the probe (or probes), and to read the fluorophore emissionduring the low detection-step temperature when the probes (either anallele-discriminating probe according to this invention or anappropriately labeled molecular beacon probe) are hybridized. We thendetermine the ratio of fluorescence of each probe to total-SYBR-signal.This ratio minimizes differences among replicate assays due todifferences in product accumulation. Because differences are minimized,such ratios can be used for end-point analysis as well.

The use of ratios of single-stranded product to double-stranded productpermitted by primers and probes according to this invention is atechnique for reducing scatter among replicate assays, as has beenstated. This is particularly important for end-point assays, which donot reveal reaction kinetics. An example is a LATE-PCR assay todistinguish homozygous samples from heterozygous samples utilizing oneprimer pair for both alleles and an allele-discriminating probeaccording to this invention. FIG. 2 illustrates the reduction in scatterachieved when applied to a LATE-PCR amplification with a low-temperaturedetection step performed with a SYBR dye (in this case SYBR Gold), anallele-discriminating probe for one allele labeled with Cy5, excitationof the dye and readings of signals from the dye (at 72° C., theextension temperature) and the fluorophore (at 55° C., a low-temperaturedetection following primer extension). Panel A presents the real-timereadings from the fluorophore for replicate homozygous samples (circle21) and replicate heterozygous samples (circle 22). As is apparent,scatter among replicates blurs the difference. Panel B, however, plotsthe ratio of Cy5 signals to SYBR signals for the homozygous samples(circle 23) and heterozygous samples (circle 24). The scatter reductionis sufficient to permit an end-point assay.

This invention also includes mismatch tolerant Low-T_(m) orSuper-Low-T_(m) linear single-stranded probes that are labeled,preferably terminally labeled, with a fluorophore excitable by emissionfrom a fluorescent DNA dye (for example, SYBR Green I or SYBR Gold) andthat are quenched to reduce background fluorescence. These probes carrya quenching moiety that suppresses fluorescence in the absence oftarget. Mismatch-tolerant linear probes have a tendency to fold and formshort double-stranded regions as the temperature is lowered. Use of alow-temperature LATE-PCR detection step exacerbates this tendency. Thisdoes not occur when the probe sequence is hybridized to the targetsequence. If the probe includes a fluorophore that is excited byemission from a SBYR dye that is present in the reaction mixture, thedye intercalates into or otherwise associates with the unintendeddouble-stranded region of the unbound probe molecules and thus excitesthe fluorophore of the probe by FRET. The result is an increase inbackground fluorescence at low temperature.

Quenching of mismatch-tolerant probes according to this invention isobtained by addition of a quenching moiety, for example, a DABCYL or aBlack Hole™ quencher (BHQ), to the probe at a location at which itquenches fluorophore fluorescence resulting from unintended secondarystructure within the unbound probe. We prefer to add the quencher at theend opposite to the fluorophore whenever possible. Example 2 belowexemplifies two possible techniques, simply adding a quencher orconstructing a quenched hairpin, that is, a specifically designedsecondary structure that brings the quencher in close proximity to thefluorophore, to the secondary structure, or both. Preferably the T_(m)of the constructed secondary structure is at least 5° C. higher than theT_(m) of any alternative secondary structure so that in the absence oftarget most probe molecules are in the hairpin configuration andbackground fluorescence is low. The T_(m) of the constructed stem isbelow the T_(m) of the probe hybridized to perfectly matched target andsimilar to the T_(m) of the probe hybridized to its mismatched targets,such that hybridization to targets of sequence within the stem is notprevented by formation of the stem.

Detection and identification of nucleic acid targets can be accomplishedby utilizing one or multiple low-temperature mismatch tolerant probesthat signal when hybridized, including mismatch-tolerant molecularbeacon probes, linear single-stranded probes that are indirectly excitedby exciting a fluorescent DNA dye, and quenched linear probes accordingto this invention. A probe mixture may, for certain embodiments, includeas well at least one allele-specific probe according to this invention.A useful technique is to utilize the ratio of fluorescence of two probesas a function of temperature to distinguish among targets having asimilar with T_(m) respect to at least one of the probes. We sometimesrefer to curves of such a ratio as a “fluorescence signature” of atarget.

With LATE-PCR that includes a low-temperature detection step it ispossible to combine the effect of detection temperature with the effectof fluorescence signature. An assay we have used with multiplemismatch-tolerant probes, including but not limited to quenched,single-stranded, indirectly excitable probes according to thisinvention, is a LATE-PCR amplification consisting of a high-temperaturestep to denature double-stranded DNA (95° C. for 2 min), followed byexponential phase amplification utilizing both Limiting Primer andExcess Primer (30 cycles of 95° C. for 10 sec, 60° C. for 15 sec, and78° C. for 40 sec), followed by the completion of the exponential phaseand the subsequent linear phase during which probe detection steps areincluded (40 cycles of 95° C. for 10 sec, 60° C. for 15 sec, 78° C. for40 sec, 55° C. for 20 sec, 50° C. for 20 sec, 45° C. for 20 sec, and 40°C. for 20 sec). This provides four detection temperatures below theprimer annealing temperature, 60° C. Double-stranded production can bemonitored by emission from SYBR dye at the primer-extension temperature,78° C., which is above the T_(m) of any probe. Fluorophore emission canbe monitored at each low-temperature from 55° C. to 40° C. Following thelast cycle, the temperature can be dropped to a low value, for example30° C. and slowly increased for melting analysis. In addition todetected fluorescence levels, ratios of fluorophore fluorescence to dyefluorescence and ratios of fluorophore fluorescence can be used togenerate amplicon-differentiating information.

Certain of the Figures are illustrative of techniques that takeadvantage of the foregoing possibilities. FIG. 4 shows the meltingbehavior of two mismatch-tolerant probes against the 16s ribosomal RNAgene of several species of Mycobacteria. Two probes were used: thehairpin-forming, quenched probe described in Example 2, having thesequence 5′-Cy5-CTG GAT AGG ACC ACG AGG CCA G-BHQ II-3′ (SEQ. ID No. 2)and a TAMRA-labeled probe having the sequence 5′-G CAT GTC TTG TGGTGG-TAMRA-3′ (SEQ. ID No. 3). It was found that the latter probe, whichwas unquenched, gave discernable signals above background for severalspecies. Panel A of FIG. 4 presents melting curves for the hairpin probewith no target (line 41), M. asiaticum (line 42), M. gordonae (line 43),M. heidelburgense (line 44), M. malmoense (line 45) and M. marinum (line46). Panel B presents melting curves for the TAMRA-labeled probe with notarget (line 47), M. asiaticum (line 48), M. gordonae (line 49), M.heidelburgense (line 50) M. malmoense (line 51), and M. marinum (line52). Panel C of FIG. 4 plots the ratio of TAMRA fluorescence to Cy 5fluorescence), M. asiaticum (line 53), M. gordonae (line 54), M.heidelburgense (line 55), M. malmoense (line 56) and M. marinum (line57).

Another analytical technique is to plot the rate of fluorescence changefrom fluorophores as a function of temperature. FIG. 5 presents suchplots for the foregoing Cy5-labeled quenched hairpin probe according tothis invention and the TAMRA-labeled unquenched probe, both describedabove. Panel A is the quenched hairpin probe, and Panel B is theTAMRA-labeled probe. The plots show melting peaks for M. asiaticum(lines 61, 71), M. gordonae (lines 62, 72), M. heidelburgense (lines 63,73), M. malmoense (lines 64, 74), and M. marinum (lines 65, 75). Usingboth probes, it is possible to distinguish the five targets by meltingpeaks. The Cy5-labeled probe by itself was able to distinguish M.gordonae (line 62) from the others. The TAMRA-labeled probe by itselfcould distinguish each of M. asiaticum (line 71), M. gordonae (line 72)and M. marinum (line 75) from one another. Taken together, the probescould distinguish M. heidelburgense from M. asiaticum, because M.heidelburgense yielded a high peak with the Cy5 probe and a low peakwith the TAMRA probe, whereas M. asiaticum yielded the opposite. With asingle probe per amplicon, relative peak heights may reflect differencesin product concentration. Here, however, both probes detect the sameamplicon, so relative peak heights reflect differences in probe-targetmelting characteristics.

Another analytical tool, described above, is to use one or morefluorescence ratios, such as, in the particular embodiment discussedhere, the ratio of TAMRA fluorescence to Cy5 fluorescence at the sametemperature or at different temperatures during the PCR. A usefulstrategy for probe design include designing one probe to bind to aconserved region common to multiple species to serve as a reference, orincluding, where needed, utilizing a portion of the Limiting Primersequence as a conserved region. This is an option for LATE-PCR, becauseprobe T_(m)'s are well below the T_(m) of the Limiting Primer and theannealing temperature, so a probe with a common sequence does notinterfere with amplification. FIG. 6 shows the results using acombination of fluorescence ratios. In this embodiment we utilized asone ratio the TAMRA/Cy5 fluorescence values each collected at the 40° C.detection temperature and as the other ratio the ratio of TAMRA/Cy5fluorescent signals collected at 45° C. and 55° C., respectively,detection temperature. FIG. 6 plots both ratios at a particular cycle,in this instance cycle 50. Six replicates yielded non-overlapping datafor the various species M. asiaticum (circle 81), M. gordonae (circle82), M. heidelburgense (circle 83), M. malmoense (circle 84), and M.marinum (circle 85).

Measuring probe fluorescence at different temperatures during PCR hasadvantages over limiting the analysis to post-PCR melts. One advantageis the ability to compare fluorescence values at a specific number ofcycles after the threshold cycle, C_(T) value, is reached. This enablesthe use of ratios with SYBR dyes (or other intercalating dyes) asdescribed above. Another advantage is that each sample has backgroundfluorescence measured at each temperature during cycles prior toamplicon detection. Thus, accurate adjustments can be made forsample-to-sample variations in background fluorescence. It is possibleto measure fluorescence at many temperatures during the PCR, providingnearly complete melting analysis over the temperature range at which aprobe shows differences in hybridization to different targets. Thenumber and duration of these steps depends in part on the capabilitiesof the detection equipment. Continuous fluorescence detection duringincreases or decreases in temperature is possible with some thermalcyclers. Detection at multiple temperatures need not begin until somepoint shortly before an initial rise in fluorescence is expected.Detection at multiple temperatures can be done every cycle, or at someother interval, for example every fifth cycle. Eliminating multipledetection steps during the initial cycles and reducing the frequency ofthose steps reduces the overall time required to complete theamplification reaction. When utilizing the ratio of probe fluorescenceto dye fluorescence, preferably probe fluorescence is measured over thetemperatures at which the probe hybridizes to its targets, and SYBRfluorescence is measured at temperatures at which probes are unbound.Most preferably, SYBR fluorescence is measured at the extensiontemperature. Since the probe fluorescence increases at cycles wellbeyond the threshold cycle (C_(T)) value while the SYBR fluorescenceplateaus, these ratios will change during the amplification reaction.Therefore, it is important to compare ratios of individual samples at aspecific number of cycles past the C_(T) value of each sample.

Analysis of single-stranded DNA products can also be accomplished usinga single mismatch-tolerant probe whose signal is measured at more thanone, for instance two or three, different temperatures. The resultingdata can then be processed as ratios using the fluorescence values attwo or more temperatures. The ratio significantly reduces signaldifferences among replicate samples and provides quantitative measure ofthe interrogated allele. FIG. 11 shows probe fluorescence levels at twotemperatures. As illustrated in FIG. 11, probe signals arising fromhybridization of the probe to the Excess Primer strand are collected ata high temperature where the probe is allele discriminating and bindsonly to the fully complementary allele, as well as at lower temperatureswhere the probe is fully mismatch-tolerant and binds to all possibleallelic variants of the target sequence. Measurement of fluorescence atthe high and low temperature and calculation of the resulting ratios canalso be carried out as an end-point assay. We refer to these assays as“Two Temperature Normalization Assays (without background correction).”They readily distinguish homozygous and heterozygous genotypes asillustrated in FIG. 11. This type of assay can be carried out asend-point homogenous LATE-PCR assays, QE-LATE-PCR assays.

FIG. 11 reports baseline-corrected fluorescence signals. As discussed inExample 5, we prefer to use raw rather than baseline-correctedfluorescence signals from the ABI 7700, as shown in FIG. 12. Baselinecorrection potentially introduces artifacts into the normalizedfluorescent ratios of individual samples, because the correction factoris sensitive to spurious fluctuations in the background fluorescencesignals use to define baseline. Raw fluorescence readings are notsubject to this artifact. Reliance on raw fluorescent signals makes theassay applicable to any PCR thermocycler with fluorimeter capabilitiesor to regular thermocyclers used in combination with atemperature-regulated fluorimeter for end-point fluorescence readings.

QE-LATE-PCR Genotyping can be further refined by constructing ratios ofsignals detected at more than two temperatures. A three-temperaturemethod for normalizing end point data is given by the following formula:Normalized Fluorescence Value=(Fs−Ft)/(Fb−Ft), where (Ft=fluorescence attop temperature), (Fb=fluorescence at bottom temperature),(Fs=fluorescence at any given third temperature). The three-temperaturemethod applied to homozygous and heterozygous genotypes of a SNP sitewithin the human p53 gene is described in Example 6 and illustrated inFIG. 13.

Pyrosequencing is a real-time, isothermal, sequencing-by-synthesismethod known in the art. It is catalyzed by four kinetically balancedenzymes: DNA polymerase, ATP sulfurylase, luciferase, and apyrase. Themethod includes a sequencing primer annealed to single-stranded DNA.Each nucleotide is dispensed and tested individually for itsincorporation into the 3′ end of the sequencing primer according to thesequence of the template DNA. A successful incorporation event isaccompanied by release of pyrophosphate (PPi) in a quantity equimolar tothe amount of nucleotide incorporated. ATP sulfurylase quantitativelyconverts the released PPi into ATP in the presence of adenosine 5′phosphosulfate. ATP then drives the luciferase-mediated conversion ofluciferin to oxyluciferin that generates visible light in amounts thatare proportional to the amount of ATP. The light is detected by a chargecoupled device (CCD) camera and displayed as a peak in a pyrogram.Unincorporated dNTP and excess ATP are continuously degraded by Apyrase.Nucleotide sequence is determined from the order of nucleotidedispensation and peak heights in the pyrogram, which are proportional tothe amounts of nucleotides incorporated.

LATE-PCR efficiently generates single-stranded DNA and thus eliminatesthe need for conventional pyrosequencing sample preparation methodsrequired to generate single-stranded templates from traditionaldouble-stranded PCR products. Use of LATE-PCR products forpyrosequencing, however, requires efficient removal of reagents leftover from the amplification reaction (dNTP, pyrophosphate, and ExcessPrimers that will interfere with the pyrosequencing chemistry. Removalof leftover reagents can be accomplished by column purification, ethanolprecipitation or any known approach of PCR product purification forremoval of dNTP, pyrophosphate and excess primers from the amplificationreaction. After cleanup, the single-stranded DNA from LATE-PCR isdirectly annealed to the sequencing primer and processed forpyrosequencing according to the manufacturer's instructions. It isimportant that LATE-PCR samples should not be heated to a temperaturethat denatures the double-stranded product generated in the reaction toguarantee that the only templates available to the sequencing primer arethe single-stranded DNA products. In fact, it may not be necessary toheat up the LATE-PCR samples for primer annealing at all since thetemplate DNA is already single-stranded.

We have combined LATE-PCR amplification with simplified clean-up methodsto prepare samples for sequencing operations. See Example 7 and FIG. 14.We have devised two methods of LATE-PCR sample preparation forPyrosequencing that do not involve physical PCR product purification andcan be performed in a single tube. In the first method, the problem ofleftover dNTPs from a LATE-PCR amplification is addressed by usinglimiting amounts of all dNTPs during the amplification such that dNTPsare depleted in the course of the reaction (but not prematurely so as tocause insufficient production of single-stranded DNA, namely the ExcessPrimer strand), which can be determined by routine experiment. Theproblem of leftover pyrophosphate from LATE-PCR is addressed by treatingthe LATE-PCR sample with an enzyme bearing a pyrophosphatase activity,for example a pyrophosphatase such as yeast pyrophosphatase, followed byheat inactivation. The Excess Primer left over from a LATE-PCRamplification should not interfere with Pyrosequencing since thematching target sequence for these primers on the 3′ end of theextension product of the Limiting Primer (the Limiting Primer strand)is: A) bound-up in a double-stranded form and therefore not easilyavailable and B) 5-20 fold less abundant than the Excess Primer strand,depending on LATE-PCR primer ratios. However, to rule out anypossibility of mispriming by the Excess Primers on PCR products at thetemperature used for Pyrosequencing, one may optionally add anoligonucleotide complementary to the Excess Primer at the start ofLATE-PCR amplification. This complementary oligonucleotide must have aT_(m) is at least 5-10° C. below the Excess Primer T_(m), for instance,by being a few nucleotides shorter than the Excess Primer at its 3′ end,and must be blocked at the 3′ end by any method known by those skilledin the art to prevent extension of the oligonucleotide by DNApolymerases (for example, by inclusion of phosphate group). Whendesigned in this fashion, the complementary oligonucleotide does notinterfere with LATE-PCR amplification but forms a stable double-strandedhybrid with the Excess Primer at the temperature used forPyrosequencing, thereby preventing the Excess Primer from misprimingother complementary sites on amplified material. Alternatively, thecomplementary oligonucleotide can have the same length or a T_(m) thatis less than 5-10° C. below that of the Excess Primer, or both, if addedafter the LATE-PCR reaction. Additionally, a 3′ blocked oligonucleotidecontaining the same sequence as the Excess Primer, with or without othermodifications to increase its T_(m) (for example extra bases at the 3′end or use of LNA analogs etc.), can be added after the LATE-PCRreaction in a concentration sufficient to out-compete Excess Primers forthe complementary site on the 3′ end of the Limiting Primer strand.

The second method includes pretreatment of LATE-PCR samples with thesame enzyme and substrate mixtures used for Pyrosequencing followed byprimer annealing and addition of individual dNTPs for Pyrosequencing. Inthis method the order of the manufacturer's recommended protocol isreversed (i.e., the normal protocol calls for primer annealing followedby addition of Pyrosequencing reaction mix). In this method, the apyrasepresent in the Pyrosequencing mix degrades dNTPs while ATP sulfurylaseand luciferase converts pyrophosphate into ATP and light. The luciferaseand luciferin contained in these solutions provide a useful system formonitoring the breakdown of PPi as well as dNTPs. Both ATP and dATPserve as substrates for luciferase, so cessation of sample light output,as detected by the CCD camera in the Pyrosequencing machine, serves as agood approximation for cleanup. If necessary for a particularpreparation, particularly if amplicons are longer than about 100 basepairs or more than about twenty base-pairs are to be sequenced, thesubstrates depleted by these reactions (adenosine 5′ phosphosulfate andluciferin) are then replenished prior to the start of DNA sequencing. Insome cases, initial treatment will require more substrate mixture thanthe manufacturer's protocol. In cases where heating and cooling isrequired for subsequent primer annealing, these reagents will bedestroyed and need to be replaced prior to Pyrosequencing.

A variation of the second method is to add a purified enzyme with adNTPase activity, for example an apyrase such as potato apyrase, and apurified enzyme with pyrophosphatase activity, for example apyrophosphatase such as yeast pyrophosphatase, followed by heatinactivation of these enzymes, primer annealing and then conventionalPyrosequencing. Once again, leftover excess primers from LATE-PCRgenerally will not interfere with Pyrosequencing but in the case thatthey do, these primers can be dealt with using the complementaryoligonucleotide strategy described above. This second method does notrequire adjustments of dNTP concentration for different LATE-PCRamplifications, and thus saves appreciable time.

Direct Pyrosequencing of LATE-PCR products requires 0.5-4 pmoles,sometimes 2-4 pmoles, of prepared single-stranded products annealed to3-15 pmoles, sometimes 10-15 pmoles, of sequencing primer depending onthe Pyrosequencing instrument used. In the second and third samplepreparation methods, it is important that the volume of added LATE-PCRsample be less than one half, sometimes less than one third, of thetotal Pyrosequencing reaction to preserve the optimal pH of thePyrosequencing mix (pH 7.5 compared to pH 8.0 or above, for example 8.3,for PCR). Alternatively, LATE-PCR products may comprise more than halfthe reaction volume if buffer concentration and pH are adjustedaccordingly. Reagents used for monitoring the various phases of aLATE-PCR amplification, such as fluorescent DNA dyes and hybridizationprobes, are compatible with Pyrosequencing and do not need to be removedexcept when a hybridization probe is designed to bind to a region to besequenced or where the Pyrosequencing primer binds. In this case, one ofthe strategies described above for blocking the Excess Primer may beemployed to block the hybridization probe. We have determined thatreagents to inhibit mispriming during amplification, disclosed in ourconcurrently filed United States Provisional patent application, titled“Reagents and Methods for Improving Reproducibility and ReducingMispriming in PCR Amplification”, are compatible with Pyrosequencingwhen the final concentration of these compounds in the Pyrosequencingreaction is 300 nM or below, preferably 200 nM or below, and thestandard DNA polymerase for Pyrosequencing is used(exonuclease-deficient Klenow DNA polymerase fragment). By utilizing aPCR sample preparation technique that permits preparation andamplification in the same chamber or container (see, for example UnitedStates patent publication US-2003-022231-A1), in combination with aLATE-PCR amplification carried out in small volumes, preferably lessthan or equal to 10 μl, for example 2-10 μl, it is possible to obtainPyrosequencing information from small groups of cells (from one to10,000 cells) in a single-tube format. According to this“Cell-to-Sequence” assay, small groups of cells (from one to 10,000cells) are prepared for amplification according to the PCR samplepreparation technique such as those described in Pierce et al. (2002)Biotechniques 32(5): 1106-1111 (see United States patent publicationUS-2003-022231-A1), subjected to LATE-PCR amplification, and processeddirectly for Pyrosequencing in a single container, well, tube orreaction chamber as described above. As demonstrated in Example 8 belowand shown in FIG. 15, the single-tube method allows for precise andaccurate genotyping, even at the single cell, single molecule level.

A general concern of enzyme-based PCR cleanup approaches forPyrosequencing is the overproduction of breakdown byproducts that maylead to feedback inhibition of enzymes during later sequencing andshorten read lengths. These include SO₄ ²⁻, oxyluciferin, inorganicphosphate (Pi), dNMPs and AMP. One way to limit the pool of Pi and dNMPsis to reduce the concentration of dNTPs used in during PCR (though, notnecessarily to the point where they are wholly consumed during thereaction as discussed above in method one). Through quantitative PCRobservations on LATE-PCR amplicons up to six hundred bases long, we havefound that dNTP concentrations can routinely be lowered to 100 nMwithout affecting amplification efficiency. Under such conditions,Pyrosequencing on enzymatically prepared LATE-PCR reactions can beaccomplished for more than fifty consecutive bases as demonstrated inExample 9, FIG. 16.

In the case of dideoxy sequencing we have developed a protocol thatincludes dilution as the only necessary treatment of LATE-PCR amplifiedproduct. Conventional dideoxy sequencing of single-stranded ampliconfrom a LATE-PCR amplification by cycle sequencing requires 50 fmoles ofthat product and a known amount of product, as capillary electrophoresisis sensitive to the amount of product. Utilizing SYBR Green Ifluorescent DNA binding dye to monitor synthesis of double-stranded DNAand a linear probe labeled with Cy5 to monitor synthesis ofsingle-stranded amplicon, one can monitor a LATE-PCR amplification,which preferably includes a mispriming-inhibiting reagent disclosed inour United States Provisional patent titled “Reagents and Methods forImproving Reproducibility and Reducing Mispriming in PCR Amplification.”None of these three additives interferes with subsequent sequencingreactions. In a LATE-PCR reaction the extent of exponentialamplification and synthesis of double-stranded product is defined by theamount of Limiting Primer and is independent of the amount of startingtemplate. The extent of single-strand production can be limited byrestricting the amount of at least one dNTP or by restricting the numberof amplification cycles, if desired.

We have determined that, for sequencing of the Excess Primer strand(i.e., the strand made from the Excess Primer in LATE-PCR) diluting theLATE-PCR amplification with water a total of at least 20-fold or morerenders the Excess Primer strand product suitable as starting materialfor dideoxy sequencing. To ensure that the amount utilized with ourcapillary sequencer contains the required minimum amount of 50 fmoles ofmaterial to be sequenced after dilution, the linear phase of theLATE-PCR reaction must yield at least 200 femtomoles (fmoles)single-stranded DNA/microliter (μl) when the concentration of limitingprimer is 25 nanomolar (nM) (25 fmoles/μl) and so about an 8-fold excessof single-stranded DNA is needed. To estimate the concentration ofsingle-stranded DNA generated by a LATE-PCR amplification, we add to theconcentration of strands present in double-stranded DNA at the end ofthe reaction (which participate in cycle sequencing, and whoseconcentration is defined by the concentration of Limiting Primer), plusthe concentration of single-stranded DNA made per cycle (we estimatethat in general each cycle of linear synthesis yields approximately 50%of theoretical product, the theoretical product being equal to theamount of double-stranded DNA in the reaction, times the number ofcycles while the reaction remains linear. If the product accumulationstops being linear in the course of the reaction as shown by flatteningof the real-time fluorescence curve for the fluorophore, the amount ofsingle-stranded DNA made during the non-linear phase is inferred fromthe fold-increase in fluorescent signals between the last cycle when thereaction was linear to the final cycle of the amplification reaction.Typically, if the concentration of single-stranded product produced in aLATE-PCR amplification is 200 fmoles/ul, we dilute the Excess Primerstrand 1:8 to 25 fmoles/ul and use 2 ul of diluted products (50 fmoles)directly into a 20 ul dideoxy sequencing reaction. Under theseconditions the total dilution factor of LATE-PCR products into thesequencing reaction is 80-fold. One can use as much as 8 μl of dilutedLATE-PCR products (200 fmoles) into the sequencing reaction for a totaldilution of 20 fold and still obtain interpretable sequencechromatograms.

Sample purification is necessary because leftover reagents from PCRamplification, such as dNTP and primers, will interfere with dideoxysequencing. LATE-PCR replaces sample preparation by ethanolprecipitation or affinity columns with a simple dilution step in water.Preparation of LATE-PCR for dideoxy sequencing only requires dilution ofexcess single-stranded DNA products in water at least 8-10 fold to aconcentration of 25 fmoles/μl, followed by addition of 50-200 fmolessingle-stranded DNA product to a dideoxy-cycle sequencing reactioncontaining 10 pmoles sequencing primer. The total dilution factor in thefinal dideoxy sequencing mix is at least 20-fold. Under theseconditions, leftover dNTPs from LATE PCR are too diluted to interferewith dideoxy sequencing. Carryover Excess Primer from LATE-PCR is alsonot a problem, because the template to which these primers bind, theLimiting Primer strand, is present at a very low concentration after thedilution step and is fully hybridized to the Excess Primer strand. Forthese two reasons the Excess Primer does not serve as a sequencingprimer. Example 10 and FIG. 17 demonstrate the effectiveness of our“dilute and go” method. FIG. 17 presents sequence chromatographsobtained using symmetric PCR and the traditional sample preparationmethod (purification of DNA products using Qiagen columns, followed byquantification by gel electrophoresis; total preparation time: 1 hr),and sequence chromatographs obtained using LATE-PCR and dilution inwater (total preparation time: 30 seconds). The sequence chromatographsare nearly identical.

Example 11 and FIGS. 18-19 illustrate strategies for LATE-PCRamplification of more than one product from the same DNA template in thesame reaction. Thus, these reactions contain two pairs of primers (eachcomprised of an Excess Primer and a Limiting Primer) that amplify twoseparate sequences within a contiguous template. The two pairs ofprimers can be arranged such that both Excess Primers and both LimitingPrimers hybridize to the same strand of the template, or to oppositestrands of the template. As one versed in the art will appreciate, whenlike primers hybridize to opposite strands of the template the twoExcess Primers can extend either “inwardly” or “outwardly” on theirrespective template stands. FIG. 19 also shows that sequences of bothExcess Primer strands can be obtained from the same reaction mixture viathe “dilute-and-go” method.

Example 12 and FIG. 20 show that the amount of ssDNA and dsDNA generatedby a LATE-PCR amplification can be measured independently and can beused to calculate the ratio ssDNA/dsDNA which, in turn, can be used todetermine whether the amount of ssDNA thus far accumulated is sufficientfor subsequent sequencing via the “dilute-and-go” method.

Example 13 and FIG. 21 show the “dilute-and-go” method employed on a50:50 mixture of LATE-PCR amplicons having two closely related, butdifferent sequences. FIG. 22 shows that mixtures comprised of 90:10 and10:90 ratios of two LATE-PCR amplicons having closely related, butdifferent sequences can be distinguished from pure 100:0 and 0:100mixtures as well as 30:70 and 70:30 mixtures via the “dilute-and-go”method. In order to accomplish this type of analysis it is necessary tocorrect the observed amplitudes of each nucleotide peak at eachheterplasmic position in terms of the expected amplitude of theequivalent “pure” nucleotide at that position. Once this is done,relative amounts of each sequence can be calculated as the ratio ofamplitudes (corrected nucleotide 1)÷(corrected nucleotide 1+correctednucleotide 2). Thus, as in the case of mitochondrial DNA sequences thatdiffer, LATE-PCR and dideoxy “dilute-and-go” methods described hereincan be used to detect heteroplasmy. The dideoxy method for measuringheteroplasmy is particularly advantageous because it can be used tosurvey many hundreds of nucleotides in a single analysis. Although notwishing to be bound by any theory, we believe that the methods describedherein work, in contrast to previous attempts based on symmetric PCR anddideoxy-sequencing, because LATE-PCR generates highly homogeneouspopulations of single-stranded amplicons. Symmetric PCR in contrasttends to generate populations of full length molecules together withsome partial amplicons and some misprimed amplicons.

Example 14 and FIG. 23 show that a LATE-PCR together with at least onesingle mismatch-tolerant probe can be used to generate end-point meltingcurves which in turn can be used to quantify the relative amounts of twoor more mixed LATE-PCR amplicons having closely related, but different,sequences. Quantitative end-point melting analysis (QE) LATE-PCR ofmixtures of related amplicons is made possible by virtue of the factthat LATE-PCR generates single-stranded products. Thus, when one or morelabeled mismatch-tolerant probes are present in the reaction, theprobe(s) hybridize first to the most complementary target sequence andthen, if the temperature is lowered sufficiently, to all related targetsequences. Thus each probe/target hybrid in the set has its own meltingtemperature and the magnitude of the melting peak derived from eachprobe/target hybrid accurately reflects the amount of each accumulatedtarget sequence. Quantitative measurements of either the amplitude, ortwo dimensional area of each melting curve can then be used to calculatethe relative abundance of each target sequence. The data shown in FIG.23 demonstrate that this method can be used with 99.7% confidence todistinguish between 0:100-10:90-50:50-90:10-100:0 mixtures of twosequences that differ by a single nucleotide.

Assays according to this invention, whether carried out in the presenceor absence of the reagent described in our U.S. Provisional patentapplication 60/619,620 can be independently optimized to avoid orminimize mispriming by adjusting the concentration of the DNApolymerase, for example Taq polymerase, added to the reaction.Decreasing mispriming by adjusting polymerase can be observed in termsof the kinetics of the LATE-PCR reaction using a probe of the ssDNA, aswell as by the composition of the final product revealed by variousmeans known in the art. We have found that it is experimentallyconvenient to start with a typical excess concentration of Taqpolymerase and then to decrease this concentration in steps. While toolittle polymerase can cause the reaction to become inefficient (manifestas a significant decrease in the rate or extent of productamplification), optimal levels of polymerase results in a LATE-PCRamplification assay with efficient dsDNA amplification and sustainedssDNA synthesis over many cycles. Example 15 demonstrates that theoptimal level of polymerase can be judged by the dsDNA signal observedusing a double-strand dye such as SYBR Green plus the melting curve ofthe dsDNA product, also observed using SYBR Green. Example 16 and FIG.24 show that when such assays are probed for a specific ssDNA productgenerated from different amounts of starting material, the resultingplots are linear and parallel over many cycles of ssDNA production.

EXAMPLES Example 1. Binding Dye Versus Binding Dye Plus Labeled Primers

To compare the performance of an intercalating dye to the performance ofthe dye used in combination with a primer that includes an interactingfluorophore, an extension assay was performed. The dye utilized was SYBRGreen I at a dilution of 1:40,000.

Three nucleotide strands were included. A DNA template, an extendableDNA primer (5′ labeled with Cy5, complementary to the template, andhaving a T_(m) of 60° C.), and a non-extendable DNA oligonucleotide (3′end blocked with a phosphate group) also complementary to the target, ata location 3′ to the primer, also labeled with Cy5 fluorophore, andhaving a higher T_(m) of 79° C. The spacing between the primer and thenon-extendable nucleotide was chosen such that primer extension productsup to the non-extendable oligonucleotide would all have T_(m)'s below79° C.

The reaction mixture for the primer extension assay included 0.5micromolar (μM) template DNA, 1.5 μM primer and 1.5 μM of thenon-extendable oligonucleotide. The mixture also included 1×PCR buffer,3 millimolar (mM) MgCl₂, 250 nanomolar (nM) of each dNTP, 1:40,000×SYBRGreen I, and Taq DNA polymerase. The reaction mixture was heated to 50°C. for 2 minutes so as to bind the primer and the non-extendableoligonucleotide, and to generate primer extension products short ofreaching the non-extendible oligonucleotide. Duplicate samples were run.

Following the primer-extension reaction, the product was subjected tomelt analysis in which the SYBR Green dye was excited as the temperaturewas changed. Fluorescence readings were taken at the wavelength of thedye's emission and at the wavelength of the fluorophore's emission asthe temperature was increased through the range of melting temperaturesencompassing the unextended primer and the non-extendableoligonucleotide. Melt curves, the first derivative of fluorescence withrespect to temperature plotted against temperature, are presented inFIG. 1, wherein Panel A presents the curves 1 for the two samples, datafrom dye emissions and Panel B presents curves 2 for the two samples,data from Cy5 emissions.

Example 2. Quenched Mismatch-Tolerant Probes

A labeled probe was designed to have a consensus sequence complementaryto the 16S ribosomal RNA gene of Mycobacterium. Secondary structure waspredicted according to the Mfold programs (Zucker, M (2003), “Mfold webserver for nucleic acid folding and hybridization prediction,” NucleicAcids Res 31: 3406-3415) with sodium concentration set at 70 millimolar(mM) and magnesium concentration set at 3 mM. The sequence of the probewas Cy5-AATACTGGATAGGACC ACG AGG (SEQ. ID No. 1), with predictedsecondary structure formed by hybridization of the underlined regions.The predicted T_(m) of the probe's secondary structure was 37° C. Thisprobe was tested in samples containing no target, M. gordonae, or M.asiaticum in mixtures containing SYBR Green I dye, wherein the dye wasexcited directly and the fluorophore was in turn excited indirectly.Results of Cy5 fluorescence versus temperature are presented in FIG. 3,Panel A. Line 31 (no target) shows high background fluorescence but line32 (M. gordonae) and line 33 (M. asiaticum) show discernable signalsabove background. To quench the background fluorescence, anon-fluorescent quencher (a Black Hole™ II quencher) was added to the 3′terminal nucleotide of the probe. The modified probe was similarlytested, and the results are shown in Panel B of FIG. 3. As can be seen,background fluorescence (line 34, no target) dropped markedly, and thesignals from M. gordonae (line 35) and M. asiaticum (line 36) were muchhigher above background.

Another technique for quenching a probe is to construct the probe tohave a hairpin structure terminally labeled with an appropriatefluorophore on one end and a quencher on the other. We constructed aprobe having the sequence Cy5-CTGGATAGGACCACGAGGCCAG-BHQII (SEQ. ID. No.2), wherein the underlined sequences are complementary and form ahairpin stem. We added the three 3′-terminal nucleotides for the purposeof achieving the stem. The predicted melting temperature of this probewith a perfectly matched target is 60° C. The predicted T_(m) of thestem is about 48° C. (based on the predicted unmodified nucleotide stemT_(m) of 40° C. not accounting for the increased affinity of thefluorophore-quencher interaction). This probe was also tested asdescribed above, and the results are presented in Panel C of FIG. 3.Background fluorescence (line 37, no target) was quite low, and thesignals from M. gordonae (line 38) and M. asiaticum (line 39) were highabove background.

Example 3. Real-Time and End-Point Genotyping Using Mismatch-TolerantProbes

This example illustrates identification of homozygous samples andheterozygous samples for the G269 allele of the human Hexosaminidase A(Hex A) gene responsible for Tay-Sachs disease using real-time LATE-PCRamplification and a Cy5-labeled, low-T_(m), mismatch-tolerant linearprobe excited indirectly by emission from a SYBR dye. Probehybridization was monitored twice during each amplification cycle withinthe detection temperature space of LATE-PCR, first at 55° C., atemperature at which the probe is allele-discriminating in this assayand binds exclusively to its perfectly matched target, and then at 40°C., a temperature at which the probe is mismatch-tolerant and binds tothe totality of alleles of its target sequence in the amplificationreaction. Detection of specific alleles and total alleles with themismatch tolerant probe permits correction of stochastic tube-to-tubevariations in amplicon yield among replicate samples. The ratio ofallele-specific-to-total alleles in the reaction (Cy5 at 55° C./Cy 5 at40° C.) allows normalization of replicate sample for end-pointgenotyping. Genotypic information is derived from the ratio values. Inthe case of homozygous samples, probe signals detected underallele-discriminating conditions are the same as probe signals detectedunder mismatch-tolerant conditions, since in both cases the probe isbinding to 100% of the target sequence alleles. In contrast, in the caseof heterozygous samples, probes signals detected underallele-discriminating conditions are half as intense as probe signalsdetected under mismatch tolerant conditions, since the probe is bindingto only 50% of the target sequence alleles under allele-discriminatingconditions but to 100% of the alleles under mismatch tolerantconditions. Hence, homozygous samples have higher Cy5 at 55° C./Cy 5 at40° C. ratios than heterozygous samples. This method of genotyping onlyrelies on detection of a single allele.

The sequences and the concentration adjusted melting temperature,T_(m[0]), of the LATE-PCR primers and the probe are as follows. TheLimiting Primer has the sequence 5′CGAGGTCATTGAATACGCACGGCTCC 3′ (SEQ.ID No. 17). It has a concentration adjusted T_(m [0]) of 63.2° C. at 25nM. The Excess Primer has the sequence 5′ TAACAAGCAGAGTCCCTCTGGT 3′(SEQ. ID No. 4). It has a concentration-adjusted T_(m[0]) of 61.8° C. at1 μM. The probe has the sequence 5′ Cy5-GGGACCAGGTAAGAA 3′ (SEQ. ID No.5). It has a T_(m) of 56.3° C. It is a Low-T_(m) probe and when usedwith a 65° C. annealing temperature, also a Super-Low-T_(m) probe.

Replicate LATE-PCR assays (n=15) were set up for each different genotype(homozygous G269 and heterozygous G269) in 1×PCR buffer, 3 mM MgCl₂, 250micromolar (μM) dNTP, 25 nM limiting primer, 1000 nM excess primer, 1.25units Taq DNA polymerase, 0.6 μM Cy5-labeled probe, and a 1:40,000dilution SYBR Gold I. PCR cycles parameters were 95° C. for 3 minutes,then 25 cycles at 95° C. for 10 sec, 65° C. for 20 sec, and 72° C. for20 sec, followed by 30 cycles at 95° C. for 10 sec, 65° C. for 20 sec,72° C. for 20 sec, 55° C. for 20 sec, and 40° C. for 20 sec withfluorescence acquisition at 55° C. and 40° C. in the Cy5 channel. FIG. 7shows analysis of the ratios of Cy5 signals at 55° C. to the Cy5 signalsat 40° C. and demonstrates that these ratios are suitable for end-pointgenotyping for any amplification cycle past the probe detectionthreshold. In this figure, homozygous samples (circle 91) have ratiosapproximately twice the ratio of heterozygous samples (circle 92).

Example 4. Analysis of Multiple Targets Using Target-Specific Probeswith Different Melting Temperatures

Multiple probes, each labeled with the same fluorophore, can be used incombination to detect and quantify different sequences along a single,longer oligonucleotide (for example, a product of asymmetric PCR,LATE-PCR, or rolling circle amplification,) or on differentoligonucleotides. The use of Low-T_(m) probes increases the specificityfor such targets, greatly reducing or eliminating signals generated frommismatched targets. One possible application of this technology isgenotyping human DNA to identify known alleles that cause geneticdisease. This example describes temperature analyses for probe designand for detection of products.

As a starting point we chose the following targets that potentiallycould be present in an amplification product: the normal sequence of thecystic fibrosis transmembrane regulator (CFTR) gene in the region thatencodes amino acid 542 of the protein; the sequence of the Delta F508mutation, the most common CFTR mutation; and the normal sequencecorresponding to the Delta F508 mutation.

We designed Low-T_(m) allele-discriminating probes for each of the threetarget sequences. The probes were low-temperature molecular beaconprobes, each labeled with the fluorophore FAM and a quencher. The threeprobes were designed to have different T_(m)'s versus their targets inmixtures containing 70 mM Tris-HCl and 3 mM MgCl₂. The “542 probe” had aT_(m) of 40° C. (predicted value 41° C. by nearest neighborcalculation); the “508 normal probe” had a T_(m) of 47° C. (predictedvalue 46° C. by nearest neighbor calculation); and the “Delta F508probe” had a T_(m) of 54° C. (predicted value 53° C. by nearest neighborcalculation). FIG. 8 presents the melting curves from which the T_(m)values were obtained. FIG. 8 shows the negative first derivative offluorescence readings as a function of temperature for the 542 probe(line 96), the DF508 probe (line 97) and the 508 normal probe (lines 98)for duplicate samples. Roughly equal peak heights were obtained by usingtarget concentrations of 1 μM, and 542 probe concentration of 2 μM. Wetested each probe against mismatched target to check allelediscrimination, and we found that fluorescence against perfect was 5-10times the fluorescence against mismatched target.

It can be seen from FIG. 8 that even small T_(m) differences would havebeen easily resolvable. From a plot such as FIG. 8, differences of 4-5°C. would be resolvable. Deconvolution utilizing software supplied withreal-time PCR thermal cyclers might permit resolution of T_(m)'sdiffering by half that amount.

Examining the negative first derivative of the fluorescence is onemethod to determine which oligonucleotide targets are present in a givensample. FIG. 9 shows such an analysis, utilizing fluorescence abovebackground. Samples containing the normal 508 target, but no Delta F508target (circle 101) have a melting peak at 54° C., indicative of thatmolecular beacon-target hybrid. Samples containing the Delta F508target, but no normal target (circle 102) have a melting peak at about47° C., indicative of hybridization to the beacon with the mutantsequence. Samples containing both of those targets (circle 103) have abroad peak over that range of temperatures, indicating fluorescence fromboth molecular beacon-target hybrids. The presence and relativeconcentration of the normal sequence at the 542 amino acid is indicatedby the presence and relative height of the melting peak at about 40° C.Samples with 542 normal target (solid line for each numbered group) havea large peak at that temperature, samples with 542 mutant targetcontaining a single nucleotide change in this region identical to thesecond most common CFTR mutation (stippled line for each numbered group)have no peak at that temperature, and samples with both 542 targets(dashed line for each numbered group) have peaks of intermediate height.The height of the peak in samples with both 542 targets is affected bythe presence of the neighboring Delta F508 melting peak.

It may not always be possible or desirable to obtain a complete meltingprofile during the course of an amplification reaction. Further analysisof the samples described above shows that a limited number of detectionsteps could provide the information required to identify the specificoligonucleotides in a mixture. Decreasing, rather than increasingtemperature can be used. Samples were heated to 70° C., and then loweredin 5° C. decrements to 30° C. with a 30 second detection at each step.Samples containing the normal 508 target but no Delta F508 target, orcontaining the Delta F508 target but no normal target could bedistinguished based on changes in fluorescence between 60° C. and 50° C.Each combination of target oligonucleotides produced a unique pattern offluorescence change. A scatter plot of the percent change influorescence increase at 55° C. vs. the percent change in fluorescenceincrease at 45° C. is shown in FIG. 10. This analysis distinguishes thecombination of targets that are present in each sample. By using thechanges in fluorescence rather than the fluorescence intensity itself,an accurate evaluation can be made even when samples differ considerablyin the total concentration of targets, as might occur in replicateamplification samples. FIG. 10 includes duplicate samples for eachcombination of normal 508 plus normal 542 targets (marks circled 111),normal 508 plus both 542 targets (112), normal 508 plus mutant 542targets (113), both 508 plus normal 542 targets (114), both 508 plusboth 542 targets (115), both 508 plus mutant 542 targets (116), Delta508 plus normal 542 targets (117), Delta 508 plus both 542 targets(118), and Delta 508 plus mutant 542 targets (119). A similar analysiscould be done using this temperature profile during each cycle orselected cycles of an amplification reaction. Several samples with DNAof known genotypes could be amplified and the detection data used toestablish an expected range of values. This would provide a method forrapid determination of genotypes from unknown samples.

Although only 3 probes were used in this example, the combined use ofmuch higher number of probes is possible. The main limitations on thetotal number of probes are the temperature range for detection and theminimum T_(m) difference between the probe-target hybrid. These are inturn dependent on the nature of the amplification reaction and thecapabilities of the equipment and deconvolution software. For example,10 different probe-target combinations could be distinguished over a 30degree temperature range if the minimum T_(m) difference fordeconvolution is 3 degrees. This number can be increased several fold byusing multiple fluorophores.

Example 5. Two Temperature Normalization with and without BackgroundCorrection

QE LATE-PCR genotyping of the rs858521 SNP was performed with unknownDNA samples and homozygous control rs858521 (CC alleles) andheterozygous control (CG alleles) using a single Cy5-labeledmismatch-tolerant probe. Amplification and detection were performedusing an ABI Prism Sequence Detector 7700 (Applied Biosystems, FosterCity, Calif., U.S.A.), which normally generates baseline-correctedfluorescent signals. For our analysis utilizing ratios, however,fluorescent signal ratios were obtained both from baseline-correctedfluorescence signals (FIG. 11) and from raw fluorescent signals (FIG.12). FIG. 11 presents the ratio of the probe's fluorescence at 50° C. toits fluorescence at 25° C. as a function of the amplification reaction'scycle number utilizing the instrument's baseline-corrected fluorescentsignals. In FIG. 11, circle 113 is replicates of the homozygous control,circle 114 is replicates of the heterozygous control, while circles 111and 112 are the unknowns. FIG. 12 presents the same results utilizingraw fluorescence signals. In FIG. 12, circle 116 is replicates of thehomozygous control, circle 117 is replicates of the heterozygouscontrol, and circle 115 is the unknowns. The use of baseline-correctedfluorescence signals for normalization resulted in ambiguous genotypingfor one sample FIG. 11, circle 112. In contrast, use of raw fluorescencesignals for normalization provided the correct genotyping for allsamples. This result demonstrates that baseline-correction in the ABIPrism 7700 Sequence Detector software can introduce artifacts thataffect signal normalization and preferably should not be used.

Example 6. Three Temperature Normalization

Replicate LATE-PCR amplification reactions containing the rs858521 SNPprimers and a single mismatch-tolerant resonsense probe were performedwith purified genomic DNA for each genotype of the rs858521 gene SNP(1800 genomes equivalent, 18 replicate reactions of each homozygous CC,heterozygous CG, and homozygous GG genotypes). The amplified productswere analyzed by melting curves, shown in FIG. 13, panel A and bynormalizing the data, as shown in Panel B and panel C. FIG. 13A shows aplot of the raw fluorescence signals collected during melting curveanalysis following LATE-PCR amplification. The probe that was utilizedwas allele-discriminating at higher temperatures but becameprogressively more mismatch tolerant as temperature was reduced. Theintrinsic variability in product yield among replicate samples precludesdiscrimination of these genotypes by raw fluorescence signals (circle131) within the temperature window of allele discrimination for thisprobe (40° C.-60° C., previously determined with syntheticoligonucleotide targets, data not shown). FIG. 13B shows the signalsfrom each sample normalized at every temperature against the signalcollected at a fully mismatch-tolerant temperature (25° C.) for thatsample. In FIG. 13B the normalized signals for the homozygous CC allelesare circle 132, the normalized signals for the heterozygous CG allelesare circle 133, and the normalized signals for the homozygous GG allelesare circle 134. As the figure shows, normalization reduces signalscatter and allows identification of each genotype within the window ofallele discrimination. Maximum separation was observed at 52° C., whichcorresponds to the Tm of the resonsense probe that was used. Althoughsignal scatter was significantly reduced in FIG. 13B compared to FIG.13A, there was still some variability in signal intensity amongreplicate samples judging from the spread in the kinetic plots. FIG. 13Cshows that the best method to eliminate this residual signal scatteringwas by normalizing the fluorescent signals at each temperature to thefluorescent signals collected at top and bottom temperatures of thewindow of allele discrimination observed in FIG. 13B where meltingcurves start to diverge (that is, 40° C. and 60° C. respectively). InFIG. 13C the normalized signals for the homozygous CC alleles are circle135, the normalized signals for the heterozygous CG alleles are circle136, and the normalized signals for the homozygous GG alleles are circle137. If Fb and Ft are the fluorescence readings towards the bottom andthe top of the temperature window of allele discrimination,respectively, and Fs is the fluorescent reading at any given temperatureduring melt analysis, then the normalized fluorescent ratios arecalculated as:Three-Temperature Normalized Fluorescence Ratio=(Fs−Ft)/(Fb−Ft)

Simultaneous normalization of the fluorescent signals at eachtemperature to the fluorescent signals at 40° C. and 60° C. within anygiven sample further reduced fluorescent signal scatter and caused thereplicate melting curves from each genotype to become very tight (seeFIG. 13C). Fluorescent ratios calculated at a single temperature,namely, the T_(m) of the probe (52° C.) normalized using the fluorescentsignals towards the top and bottom temperatures of window of allelediscrimination (i.e., at 60° C., 40° C.) uniquely define each genotypewith greater than 99.7% certainty (i.e., error boxes consisting ofthree-standard deviations encompassing 99.7% of all possible fluorescentratios for each genotype are well separated from each other, data notshown). Similarly improved results were obtained for the rs2270517 SNPsite when fluorescent signals were calculated at the T_(m) of the probe(57° C.) normalized to the corresponding top and bottom temperatures ofwindow of allele discrimination (i.e., at 71° C., 45° C.).

Example 7. Direct Pyrosequencing of LATE-PCR Product

Replicate LATE-PCR amplifications were carried out in 25 Tl volumeconsisting of 1×PCR buffer, 3 mM MgCl₂, 20 nanomolar (nM) dNTP, 25 nMLimiting Primer, 1000 nM Excess Primer, 1.25 units Platinum Taq DNApolymerase, and 100 genomes human DNA. The sequence of the LimitingPrimer was 5′ CCGCCCTTCTCTCTGCCCCCTGGT 3′ (SEQ. ID No. 6) and thesequence of the Excess Primer was 5′ GCCAGGGGTTCCACTACGTAGA 3′ (SEQ. IDNo. 7). These sequences amplify a 94 base-pair segment from exon 11 ofthe human Hexosaminidase A gene. For LATE-PCR amplification, the thermalcycle profile was 95° C. for 3 min followed by 10 cycles of 95° C. for10 sec, and 72° C. for 20 sec, followed by 55 cycles of 95° C. for 10sec, 67° C. for 20 sec, and 72° C. for 20 sec. After the reaction 16.6μl (the equivalent of 3 pmoles of single-stranded DNA (ssDNA) asestimated empirically from previous pyrosequencing experiments) weremixed with 20 microliter (μl) 10 mM Tris-Cl pH 8.5 and placed in a wellof a microtiter plate used for pyrosequencing. For removal ofcarried-over dNTP and pyrophosphate from the LATE-PCR-amplified product,standard pyrosequencing enzyme mixture consisting ofexonuclease-deficient Klenow DNA polymerase, apyrase, luciferase, ATPsulfurylase and standard pyrosequencing Substrate Mixture consisting ofluciferin and adenosine 5′ phosphosulfate as provided in the PSQ 96 SNPReagent Kit (Pyrosequencing, Inc, Westboro, Mass.) were dispensedsequentially into the well containing the LATE-PCR sample using a PSQ 96instrument (Pyrosequencing, Inc., Westboro, Mass.) according to themanufacturer's instructions and incubated for 60 sec at 37° C. Thesubsequent dNTP additions normally carried out automatically by the PSQ96 instrument were replaced by a single addition of 10 mM Tris-Cl pH 7.5using the default volume programmed in the instrument. Following thisstep, the well containing the LATE-PCR sample received 2.5 μl 10 μMsequencing primer (5′ CTGGTACCTGAACCGTAT 3′) (SEQ. ID No. 8). Takinginto account the volume of pyrosequencing enzyme and substrate mixturesadded to the LATE-PCR sample, the final concentration of sequencingprimer was estimated to be 0.5 ™ and the final volume 50 μl. The samplewith the sequencing primer was returned to the PSQ 96 instrument againand processed according to the manufacturer's instructions except thatthe pyrosequencing enzyme and substrate additions normally carried outby the instrument were replaced by addition of similar volumes of 10 mMTris-Cl pH 7.5 followed by addition of dNTP. The resulting pyrogram isshown in FIG. 14, Panel A, which shows light signal resulting fromincorporation of particular nucleotides. The height of the peakscorresponds to the number of nucleotides incorporated during eachaddition. Referring to Panel C of FIG. 14, one sees that one of each ofthe first two nucleotides (A, T) was incorporated into the template,followed by two of the next nucleotide (C, C), and so on. Based on theheight of the peaks and the order of nucleotide additions a sequence wasderived: 5′ ATCCTATGGCCC3′ (SEQ. ID No. 9) and subsequently confirmedusing the GenBank sequence for the human Hexosaminidase A gene (GenBankaccession number: S62068). These results demonstrate pretreatment ofLATE-PCR samples with the enzyme and substrates mixtures used forpyrosequencing permits direct pyrosequencing of LATE-PCR-amplifiedproduct following primer annealing and iterative dNTP addition. Alteringthe above protocol to follow the manufacturer's instructions (i.e.,performing primer annealing followed by addition of the pyrosequencingenzyme and substrate mixtures) resulted in 80% false positive peaks uponaddition of dNTP that were not supposed to be incorporated on thetemplate. These false positive peaks were due to partial extension thesequencing primer from the leftover dNTP from LATE-PCR amplificationprior to pyrosequencing.

In a separate experiment, the same LATE-PCR sample described above wassubjected to purification using a QIAquick PCR purification kit (Qiagen,Valencia, Calif.) according to the manufacturer's instructions andrecovered at 0.375 pmoles/μl in 10 mM Tris-Cl pH. 7.5. Eight microliters(μl) of this solution (3 pmoles total) were mixed with the sequencingprimer described above to a final concentration of sequencing primer of0.5 μM in a final volume of 50 μl in 10 mM Tris-Cl pH. 7.5. The samplewas subjected to pyrosequencing using the PSQ 96 instrument according tothe manufacturer's instructions. The resulting pyrogram is shown in FIG.14, panel B. Traditional preparation, while more time-consuming andexpensive, did not give superior data as compared to our method thatproduced Panel A.

Example 8. Direct Pyrosequencing of LATE-PCR Products

To genotype single cells, replicate LATE-PCR amplifications were carriedout in a 25 μL, volume consisting of 1×PCR buffer, 3 mM MgCl₂, 100 μMdNTP, 100 nM Limiting Primer, 1000 nM Excess Primer, 1.25 units AmpliTaqGold DNA polymerase (Applied Biosystems, USA). Each reaction wasinitiated with a single human lymphoblast prepared as described inPierce et al. (2002) Biotechniques 32(5): 1106-1111 (see United Statespatent publication US-2003-022231-A1) with one of the three possiblegenotypes for the IVS-110 mutation. The sequence of the Limiting Primerwas 5′ GGCCATCACTAAAGGCACCGAGCACT 3′ (SEQ. ID NO. 10) and the sequenceof the Excess Primer was 5′ GGGTTTCTGATACGCACTGACTCTCTC 3′ (SEQ. ID NO.11). These sequences amplify a 191 base-pair segment from the β-Globingene on human chromosome 11p. For LATE-PCR amplification, the thermalcycle profile was 95° C. for 10 min followed by 65 cycles of 95° C. for10 sec, 66° C. for 15 sec and 72° C. for 20 sec. After amplification, 5μl were mixed with 6.64 μl 20 mM Tris-Acetate pH 7.6 and placed in awell of an optical plate used for Pyrosequencing. For removal ofcarried-over dNTPs and PPi from the product of LATE-PCR amplification astandard volume of Pyrosequencing enzyme mixture (consisting ofexonuclease-deficient Klenow DNA polymerase, apyrase, luciferase, ATPsulfurylase) and approximately twice the standard volume of substratemixture (consisting of luciferin and adenosine 5′ phosphosulfate) asprovided in the Pyro Gold Reagent Kit (Biotage AB, Uppsala, Sweden) weredispensed sequentially into the wells containing the LATE-PCR samplesusing a PSQ HS 96A instrument (Biotage AB, Uppsala, Sweden) using thefollowing instrument settings: enzyme mix pulse time: 23.5 ms; substratemix pulse time: 44.0 ms; reagent dispensation pressure: 400 mbar.Samples were incubated for 60 sec at 28° C. until light output droppedbelow background. Following this, 0.36 μL of a 10 μM sequencing primer:5′ GACCACCAGCAGCCTAAG 3′ (SEQ. ID NO. 12) was added to each sample for atotal reaction volume of 120 and then annealed at 80° C. for 2 minfollowed by cooling to room temperature for 10 min. In addition, a 900μM concentration of a 3′ phosphorylated version of the LATE-PCR LimitingPrimer (SEQ. ID NO. 7) was also added here to prevent the 3′ end of thetemplate strand from folding over on itself and extending. Samples withthe sequencing primer were then returned to the PSQ HS 96A instrumentagain and processed according to the manufacturer's instructions,including normal enzyme and substrate mix additions. The resultingPyrograms from cells with a homozygous wild-type, heterozygous andhomozygous mutant genotypes are shown in FIG. 15, Panels A-C,respectively. Light units and peak heights are as explained in Example7. The relative height of the peaks corresponds to the number ofnucleotides incorporated at each position. Referring to panel A of FIG.15, one sees that the second peak (T) is half as tall as the first peak(G), one third as tall as the third peak (G), one forth as tall as thefourth peak (A) and the same height as peaks 5-8 (TAGA). The sequencefor the first eight peaks is thus read as: GGTGGGAAAATAGA (SEQ. ID No.13). Based on the height of the peaks and the order of nucleotideadditions, the wild-type β-Globin sequence in FIG. 15, panel A wasderived and subsequently confirmed using the GenBank sequence for thehuman β-Globin Gene. A heterozygous (Panel B) or homozygous (Panel C)mutation was confirmed at the IVS-110 site, indicated by arrows. It isof note in Panel B that the 1.5 unit “C” peak followed by a 0.5 unit “T”peak indicates a “C” base in both alleles followed by a “C” in oneallele and a “T” in the other allele. These results demonstrate thatpretreatment of LATE-PCR samples with the enzyme and substrates mixturesused for Pyrosequencing permits direct Pyrosequencing of LATE-PCRfollowing primer annealing and iterative dNTP additions. Altering theabove protocol to follow the manufacturer's instructions (i.e.,performing primer annealing followed by addition of the Pyrosequencingenzyme and substrate mixtures) resulted in 80% false positive peaks uponaddition of dNTP that were not supposed to be incorporated on thetemplate. These false positive peaks were due to partial extension ofthe sequencing primer with leftover dNTPs.

Example 9. Pyrosequencing of LATE-PCR Products for Long Sequences

A LATE-PCR amplification was carried out in a 25 μl volume consisting of1×PCR buffer, 3 mM MgCl₂, 100 μM dNTP, 100 nM Limiting Primer, 1000 nMExcess Primer, 1.25 units AmpliTaq Gold DNA polymerase (AppliedBiosystems, USA) and 50 nM of mispriming-reducing reagent 9-22DD asdisclosed in our filed United States Provisional patent application,titled “Reagents and Methods for Improving Reproducibility and ReducingMispriming in PCR Amplification”. Reagent 9-22DD is a hairpinoligonucleotide having a stem nine nucleotides long and asingle-stranded loop 22 nucleotides long. The oligonucleotide ismodified by the addition of 5′ terminal and 3′ terminal Dabcyl moieties.Its nucleotide sequence is 5′ CGCGGCGTCAGGCATATAGGATACCGGGACAGACGCCGCG3′ (SEQ. ID. No 14). The reaction was initiated with 20 genomeequivalents of human DNA. The sequence of the Limiting Primer was 5′GGTCAGCGCCGGGCTGCAAGTGTAGA 3′ (SEQ. ID NO. 15) and the sequence of theExcess Primer was 5′ GATGGGTGGAGCTTGTCTTGAGG 3′ (SEQ. ID NO. 16). Thesesequences amplify a 78 base-pair segment near the p53 gene on humanchromosome 17p. For LATE-PCR amplification, the thermal cycle profilewas 95° C. for 10 min followed by 60 cycles of 95° C. for 10 sec, 66° C.for 10 sec and 72° C. for 20 sec. After amplification, 7.5 μl of productwas mixed with 9.96 μl 20 mM Tris-Acetate pH 7.6 and placed in a well ofan optical plate used for Pyrosequencing. For removal of carried-overdNTPs and PPi from LATE-PCR a standard volume of Pyrosequencing enzymemixture (consisting of exonuclease-deficient Klenow DNA polymerase,apyrase, luciferase, ATP sulfurylase) and approximately twice thestandard volume of substrate mixture (consisting of luciferin andadenosine 5′ phosphosulfate) as provided in the Pyro Gold Reagent Kit(Biotage AB, Uppsala, Sweden) was dispensed sequentially into the wellcontaining the LATE-PCR samples using a PSQ HS 96A instrument (BiotageAB, Uppsala, Sweden) using the following instrument settings: enzyme mixpulse time: 23.5 ms; substrate mix pulse time: 44.0 ms; reagentdispensation pressure: 400 mbar. The sample was then incubated for 60sec at 28° C. until light output dropped below background. In thisamplicon, the Limiting LATE-PCR primer (SEQ. ID NO. 10) was used as thePyrosequencing primer and 0.54 μl of 10 μM solution of this was added toeach sample for a total reaction volume of 18 μl and then annealed at80° C. for 2 min followed by cooling to room temperature for 10 min.Samples with the sequencing primer were then returned to the PSQ HS 96Ainstrument again and processed according to the manufacturer'sinstructions, including normal enzyme and substrate mix additions. Theresulting Pyrogram is shown in FIG. 16. The relative height of the peakscorresponds to the number of nucleotides incorporated at each positionas described in Example 8. The correctly matching expected sequence, asdetermined from the GenBank database, is noted above the peaks withsubscripts indicating the number a given base in a row (i.e.G₁C₁A₁G₂=GCAGG). These results demonstrate that pretreatment of LATE-PCRsamples with the enzyme and substrates mixtures used for Pyrosequencingallows for reads more than fifty base pairs long.

Example 10. Direct Dideoxy Sequencing of LATE-PCR Product

PCR amplifications were performed utilizing an ABI Prism SequenceDetector 7700 (Applied Biosystems, Foster City, Calif., U.S.A.) toamplify a segment of exon 7 of the human Hexosaminidase A genecontaining the G269 mutation, which is responsible for Tay-SachsDisease. The sequence corresponds to GenBank accession number M16417.One amplification was a LATE-PCR amplification, and the product wassubjected directly to dideoxy sequencing. As a control the primerconcentrations were changed to equimolar, a conventional symmetric PCRamplification was performed, and amplified product was subjected toconventional purification prior to dideoxy sequencing.

Amplification Reaction Mixtures (Final Concentrations)

Volume: 25 μl

1×PCR buffer (Invitrogen, Carlsbad, Calif., U.S.A.)

3 mM MgCl₂

10 μM dNTPs

0.6 μM Probe (LATE-PCR only)

1:41,666 dilution SYBR Gold Dye (Molecular Probes, Eugene, Oreg., U.S.A)

1.25 Units Platinum Taq DNA polymerase (Invitrogen)

6 ng human genomic DNA (equivalent to 1000 genomes)

Primers: for LATE-PCR, 25 nM Limiting Primer and

-   -   1000 nM Excess Primer; (for the control, 300 nM of each of the        same primers).        Oligonucleotide Sequences

Limiting Primer: (SEQ. ID. No. 17) 5′ CGAGGTCATTGAATACGCACGGCTCC 3′Excess Primer: (SEQ. ID. No. 18) 5′ TAACAAGCAGAGTCCCTCTGGT 3′ Probe:(SEQ. ID No. 19) 5′ Cy5 GGGACCAGGTAAGAA- Phosphate 3′Cycle Sequencing Reaction Mixture

Volume: 20 μl

-   -   100 femtomoles (fmoles) product being sequenced    -   5 picomoles (pmoles) Sequencing Primer (either the Limiting        Primer or the Excess Primer)    -   1×DTC5 Quick Start Master Mix (Beckman Coulter, Inc., Fullerton,        Calif., U.S.A.)    -   [includes dNTPs, ddNTP, buffer, MgCl₂].        Dideoxy Sequencing

Sequencing reaction mixtures were subjected to cycle sequencing andcapillary electrophoresis in a CEQ 2000XL DNA Sequence (Beckman Coulter,Inc., Fullerton, Calif., U.S.A.) using the CEQ 2000 Due TerminationCycle Sequencing Kit (Beckman Coulter) according to the manufacturer'sinstructions.

LATE-PCR Amplification and Sequencing Preparation

The LATE-PCR amplification reaction mixture was subjected to thermalcycling as follows: 95° C. for 3 min; 20 cycles of 95° C. for 10 sec,65° C. for 20 sec and 72° C. for 20 sec, and 70 cycles of 95° C. for 10sec, 65° C. for 20 sec, 72° C. for 20 sec, 55° C. for 20 sec and 40° C.for 20 sec. Synthesis of double-stranded amplicon was monitored byexciting the SYBR dye and reading its fluorescence during the 72° C.primer-extension step. Synthesis of single-stranded product followingexhaustion of the Limiting Primer was monitored by exciting the SYBR dyeand reading fluorescence from the low-T_(m) Probe's Cy5 fluorophoreduring the 40° C. low-temperature detection step.

To obtain 100 fmoles of the extension product of the Excess Primer,dilution of the amplification product was necessary. We estimated theamount of product in the 25 ul of reaction product in the followingmanner. First, the amount of that product in double-stranded productmade during the initial amplification cycles is dictated by the amountof Limiting Primer. In this example that was 25 nM, which translates to25 fmoles/μl. The concentration of single-stranded extension productmade during the linear phase of LATE-PCR amplification, that is, afterexhaustion of the Limiting Primer, was estimated by dividing that phaseinto two parts determined by inspection of the Cy5 fluorescence curve: afirst part in which amplification proceeds arithmetically, and a secondpart in which product accumulation has slowed. For the first part, whichin this example was six cycles, we assumed an amplification efficiencyof 50%, based on Gyllensten, U. B. H. and Erlich, A. (1988), “Generationof Single-Stranded DNA by the Polymerase Chain Reaction and itsApplication to Direct Sequencing of the HLA-DQA LOCUS,” Proc. Natl.Acad. Sci. USA 85: 7652-7656. Production of single strands during thesix cycles was calculated as the starting concentration (25 fmoles/μl)times the number of cycles (6) times the efficiency (0.5). Furtherproduction was estimated as the percentage increase in Cy5 signal duringthe remainder of the reaction, which in this case was 233.3%. Totalproduction during the linear phase was thus 175 fmoles/μl(25×6×0.5×2.333), and the total concentration of that product, including25 fmoles/μl in double-stranded amplicon, was estimated to be 200fmoles/μl. To obtain 100 fmoles in the cycle-sequencing reactionmixture, we diluted the amplification product 1:8 with water and used 4μl of the diluted product in the 20 μl reaction mixture. As will beappreciated, this meant that the amplification product was ultimatelydiluted 1:40.

To obtain 100 fmoles of the extension product of the Limiting Primer,our starting point was that the product of the amplification reactioncontained 25 nM of that product, or 25 fmoles/μl. We simply used 4 μl ofthe amplification product in the 20 μl cycle-sequencing reaction mixtureto obtain the desired starting amount of 100 fmoles.

Control Amplification and Sequencing Preparation.

The amplification reaction mixture was subjected to the same thermalcycling profile, except that only 18 (rather than 70) of thefive-temperature cycles were carried out, because a real-time plot ofthe intercalating dye signal indicated that the amplification plateauedat this point and only desired amplification product was made to thatpoint. The amplification products in the amplification mixture at theend of amplification were purified in conventional manner using QUIAquick PCR purification kit (Qiagen, Valencia, Calif., U.S.A.) accordingto the manufacturer's instructions. Purified amplicons were quantifiedby gel electrophoresis in a 3% agarose gel in 0.5×TBE against differentknown amounts of ΦX174 Hind III DNA markers following visualization byethidium bromide staining (0.5 Tg/ml). A volume containing 100 fmoleswas used in the cycle-sequencing reaction mixture with each sequencingprimer.

Results

The LATE-PCR and control methods both produced sequences correspondingto Genbank sequence information (accession number M 16417). FIG. 17includes four chromatographs obtained from dideoxy sequencing. Panel Ais from the LATE-PCR method with cycle sequencing utilizing the LimitingPrimer as the sequencing primer. Panel B is from the LATE-PCR methodwith cycle sequencing utilizing the Excess Primer as the sequencingprimer. Panel C is the control method utilizing the Excess Primer as thesequencing primer. Panel D is the control method utilizing the LimitingPrimer as the sequencing primer. Each chromatograph includes thefluorescence curves obtained from the labeled dideoxy nucleotides andthe nucleotide sequence determined.

Example 11. Strategies for LATE-PCR Amplification of More than OneProduct from the Same DNA Template in the Same Reaction

PCR amplifications were performed utilizing an ABI Prism SequenceDetector 7700 (Applied Biosystems, Foster City, Calif., U.S.A.) toamplify two amplicons of 549 and 464 bases designated as HV1 and HV2 Hand L strands in the same duplex reaction within the d-loop region ofHuman mitochondrial DNA based on which sequences were amplified using anExcess Primer.

Amplification Reaction Mixtures (Final Concentrations)

Volume: 25 Tl

1×PCR buffer (Invitrogen, Carlsbad, Calif., U.S.A.)

3 mM MgCl2 (Invitrogen)

250 μM dNTPs (Promega)

1.0 μM Probe (LATE-PCR only)

10× dilution SYBR Green Dye (FMC Bioproducts, Rockland Me., U.S.A)

1.25 Units Platinum Taq DNA polymerase (Invitrogen)

Human blood lymphocyte genomic DNA (equivalent to 100 mtDNA genomes)

Primers: for LATE-PCR, 50 nM Limiting Primer and 1000 nM Excess Primer.

Oligonucleotide Sequences

Probe: (SEQ. ID No. 20) 5′ Cy5- TGCTAATGGTGGAG -Phosphate 3′ HV1-HLimiting Primer: (SEQ. ID. No. 21) 5′ GCCCGGAGCGAGGAGAGTAGCACTCTTG 3′Excess Primer: (SEQ. ID. No. 22) 5′ CACCAGTCTTGTAAACCGGAGATGAA 3′ HV2-HLimiting Primer: (SEQ. ID. No. 23) 5′GTATGGGAGTGGGAGGGGAAAATAATGTGTTAG 3′ Excess Primer: (SEQ. ID. No. 24) 5′AGGTCTATCACCCTATTAACCACTCA3′ HV1-L Limiting Primer: (SEQ. ID. No. 25) 5′CACCAGTCTTGTAAACCGGAGATGAAAACC 3′ Excess Primer: (SEQ. ID. No. 26) 5′CGAGGAGAGTAGCACTCTT3′ HV2-L Limiting Primer: (SEQ. ID. No. 27) 5′AGGTCTATCACCCTATTAACCACTCACGGG 3′ Excess Primer: (SEQ. ID. No. 28) 5′GGAGGGGAAAATAATGTGTTAGT 3′Cycle Sequencing Reaction Mixture

Volume: 25 μl

100 fmoles product being sequenced

5 pmoles Sequencing Primer (either the Limiting Primer or the ExcessPrimer)

1×DTC5 Quick Start Master Mix (Beckman Coulter, Inc., Fullerton, Calif.,U.S.A.) [includes dNTPs, ddNTP, buffer, MgCl2].

Dideoxy Sequencing

Sequencing reaction mixtures were subjected to cycle sequencing andcapillary electrophoresis in a CEQ 2000XL DNA Sequence (Beckman Coulter,Inc., Fullerton, Calif., U.S.A.) using the CEQ 2000 Dye TerminationCycle Sequencing Kit (Beckman Coulter) according to the manufacturer'sinstructions.

LATE-PCR Amplification and Sequencing Preparation

The LATE-PCR amplification reaction mixture was subjected to thermalcycling as follows: 95° C. for 3 min; 15 cycles of 95° C. for 15 sec,64° C. for 10 sec and 72° C. for 45 sec, and 50 cycles of 95° C. for 15sec, 64° C. for 10 sec, 72° C. for 45 sec, and for HV1-H only 50° C. for20 sec. Synthesis of double-stranded amplicon was monitored by excitingthe SYBR Green dye and reading its fluorescence during the 72° C.primer-extension step. Synthesis of single-stranded product followingexhaustion of the Limiting Primer was monitored by exciting the SYBR dyeand reading fluorescence from the low-Tm Probe's Cy5 fluorophore duringthe 50° C. low-temperature detection step for HV1-H region only.

To obtain 100 fmoles of the extension product of the Excess Primer,dilution of the amplification product was necessary. We estimated theamount of product in the 25 μl of reaction product in the followingmanner. First, the amount of that product in double-stranded productmade during the initial amplification cycles is dictated by the amountof Limiting Primer. In this example that was 50 nM, which translates to50 fmoles/μl. The concentration of single-stranded extension productmade during the linear phase of LATE-PCR amplification, that is, afterexhaustion of the Limiting Primer, was estimated by dividing that phaseinto two parts determined by inspection of the Cy5 fluorescence curve: afirst part in which amplification proceeds arithmetically, and a secondpart in which product accumulation has slowed. For the first part, whichin this example was eleven cycles, we assumed an amplificationefficiency of 50%, based on Gyllensten, U. B. H. and Erlich, A. (1988),“Generation of Single-Stranded DNA by the Polymerase Chain Reaction andits Application to Direct Sequencing of the HLA-DQA LOCUS,” Proc. Natl.Acad. Sci. USA 85: 7652-7656. Production of single strands during theeleven cycles was calculated as the starting concentration (50fmoles/μl) times the number of cycles 11) times the efficiency (0.5).Further production was estimated as the percentage increase in Cy5signal during the remainder of the reaction, which in this case was100%. Total production during the linear phase was thus 275 fmoles/μl(50×11×0.5×1.0), and the total concentration of that product, including50 fmoles/μl in double-stranded amplicon, was estimated to be 325fmoles/μl. To obtain 100 fmoles in the cycle-sequencing reactionmixture, we diluted the amplification product 1:13 with water and used 4μl of the diluted product in the 25 μl reaction mixture.

Results

There are four possible combinations are: 1) HV1-H with HV2-H, 2) HV1-Lwith HV2-L, 3) HV1-H with HV2-L, 4) HV1-L with HV2-H. FIG. 18 shows a 4%agarose gel from electrophoresis of no-template controls (NTC), leftthree lanes; amplicons from reactions begun with 100 copies of genomicDNA, next three lanes; and in the far right lane a 100 base-pair ladder.FIG. 18 shows the formation of the HV1-H and HV2-H dsDNA amplicons of549 and 464 base pairs using 100 copies of genomic DNA at the start ofthe reaction. No template controls, NTC, did not amplify.

As one versed in the art will understand, in amplifying twosingle-stranded amplicons in the same reaction from a single template,the two excess primer strands can be generated from the same strand ofDNA or from complementary strands of DNA. We have successfully employedboth approaches. In the combinations HV1-H with HV2-H and HV1-L withHV2-L both amplicons are generated from the same DNA template strand. Inthe combinations HV1-H with HV2-L and HV1-L with HV2-H the two ampliconsare generated from complementary strands of DNA. FIG. 19 A displayssequence information for amplicon HV1-H in the duplex HV1-H with HV2-Hin the region of bases 16209-16169. FIG. 19 B displays sequenceinformation for the amplicon HV2-H in the duplex HV1-H with HV2-H in theregion bases 289-326. FIG. 19 C displays sequence information for theHV1-H amplicon in the duplex HV1-H with HV2-L in the region bases16209-16169. FIG. 19 D displays sequence information for the HV2-Lamplicon in the duplex HV1-H with HV2-L in the region bases 289-326. TheLATE-PCR produced sequences corresponding to GenBank sequenceinformation.

Example 12. Determining ssDNA Need

The amount of single stranded DNA and double stranded DNA generated by aLATE-PCR amplification can be used to determine amount of ssDNA neededfor “dilute-and-go” Dideoxy Sequencing. PCR amplifications wereperformed utilizing an ABI Prism Sequence Detector 7700 (AppliedBiosystems, Foster City, Calif., U.S.A.) to amplify the 549 baseamplicon designated as HV1 H within the d-loop region of humanmitochondrial DNA. MtDNA was extracted under lysis conditions (asdescribed in Peirce et al. (2002) Biotechniques 32(5); 1106-1111 withthe inclusion of 4 ul DTT in 100 ul of the lysis reaction mixture) froma human hair shaft. All amplifications were LATE-PCR amplifications, andthe product was subjected directly to dideoxy sequencing.

Amplification Reaction Mixtures (Final Concentrations)

Volume: 25 μl

1×PCR buffer (Invitrogen, Carlsbad, Calif., U.S.A.)

3 mM MgCl2 (Invitrogen)

250 μM dNTPs (Promega)

1.0 μM Probe (LATE-PCR only)

10× dilution SYBR Green Dye (FMC Bioproducts, Rockland Me., U.S.A)

1.25 Units Platinum Taq DNA polymerase (Invitrogen)

1 μl DNA Lysis solution (equivalent to ˜10 mtDNA genomes)

Primers: for LATE-PCR, 50 nM Limiting Primer and

-   -   1000 nM Excess Primer.        Oligonucleotide Sequences

HV1H: Limiting Primer, Excess Primer and Probe as in Example 11.

Cycle Sequencing Reaction Mixture

As in Example 11.

Dideoxy Sequencing

As in Example 11.

LATE-PCR Amplification and Sequencing Preparation

As in Example 11. The raw fluorescent data of the both CY5 and SYBRGreen were used to determine the amount of product available for asequencing reaction. The CY5/SYBR Green ratio was used to normalize allfluctuations in the raw data.

Results

Fluorescence data from the LATE-PCR amplifications is presented in FIG.20, panels A and B. FIG. 20A, e.g., line 201 shows all of the hair shaftdata plotted against amplification cycle numbers as the ratioss-DNA/ds-DNA (probe signal to dye signal). This method of analysisminimizes the variation due to when exponential amplification begins, orat what level it plateaus, and demonstrates that the efficiency ofss-DNA amplification is virtually the same in all samples except the onethat began very late. FIG. 20B shows a method for monitoring a set ofLATE-PCR assay in order to establish their readiness for dilute-and-gosequencing. The plot shows the calculated ratios ssDNA/dsDNA (probesignal to dye signal versus dye signal) for all amplified samples atcycle 45 (squares) and cycle 65 (diamonds). Only the samples that haveratios of between 0.06 and 0.10 and SYBR values between 300 and 600(those in the box) are ready for sequencing. FIG. 20B extends the use ofQuantitative End-point analysis (QE LATE-PCR) to demonstrate that after65 cycles all but one sample had accumulated sufficient ss-DNA for usein “dilute-and-go” sequencing.

Example 13. Amplicons Having Multiple SNPs

The sensitivity of the LATE-PCR and “dilute-and-go” sequencing methodcan distinguish a mixture of amplicons having multiple SNPs to the 10%resolution level. PCR amplifications were from a 2 mm human hair shaftor a single human thumbprint adhered to a glass slide. Allamplifications were LATE-PCR amplifications, and the product wassubjected directly to dideoxy sequencing. Final amplification reactionmixtures, Oligonucleotide Sequences (HV1-H), Cycle Sequencing ReactionMixture, and Dideoxy Sequencing, and LATE-PCR Amplification andSequencing Preparation were all as in Example 11.

Mixtures from 10:90 to 90:10 of the single-stranded LATE-PCR products ofeach of the three reactions were sequenced using the ‘dilute-and-go”dideoxy protocol described previously. The results are shown in FIG. 21and FIG. 22.

FIG. 21 show a 10 base segment surrounding bases 16320 and 16311 of the50:50 mixture of Human blood lymphocyte and the Human thumbprint. Thepeak heights reflect the actual 100% heights in the dideoxy sequence andnot the expected equal heights of a 50:50 mixture. Line 211 shows thepeak for the G base at this sequence and line 202 shows the peak for theA base at the same position in the sequence. Peak 212 is higher thanpeak 211 in a 50:50 mixture of human blood lymphocyte and human hairshaft having different genetic sequences, because of the fluormetriccharacteristics of dideoxy sequencing as is demonstrable by analysis ofpure sequences for the same region.

FIG. 22 shows the reciprocal percentages (90:10, 70:30, 50:50, 30:70 and10:90) of two samples at each of five SNPs locations. Sample 1 came froma Human Hair Shaft and Sample 2 came from a Human Thumbprint fromanother individual. The heights of each peak at each position weremeasured from the printouts of the dideoxy sequences and were thenscaled based on the same base of a 100% Sample 1 or 100% Sample 2control. In FIG. 22, line 222 is the intended percentage of Sample 1 inthe mixture plotted against the intended percentage of Sample 2 in themixture. Line 221 is a line fitted to the actual results, that is, theobserved percentage of Sample 1 in the mixture plotted against theintended percentage of Sample 2. The observed percentage for eachintended percentage of Sample 2 is five points, one for each base. Thedata demonstrate that there is very little scatter among the differentbases at each percentage, but the data also show that line 221 of theobserved values does not fall on top of the line of the predicted values(line 222), probably because amount of Sample 1 and Sample 2 in themixture were not exactly equal.

Example 14. Distinction of Mixtures

To distinguish samples consisting of 100% heterozygous genomic DNA fromsamples consisting of 90% heterozygous DNA and 10% homozygous genomicDNA for a single nucleotide change, we first created a DNA mixtureconsisting of 90% heterozygous DNA for the SNP site rs858521 located inhuman chromosome 17 (C/G alleles) plus 10% homozygous DNA for the sameSNP site (C/C alleles). The SNP site is listed in the NCBI dbSNPdatabase accessible through ncbi.nlm.nih.gov/entrez/query.fcgi?DB=SNP.This DNA mixture was prepared by mixing matched concentrations of thecorresponding heterozygous and homozygous DNAs provided by the ReidLaboratory at the University of Washington in Seattle. DNAconcentrations for each genomic DNA for mixing purposes were estimatedbased on the Ct values of SYBR fluorescence derived from real-timeanalysis of LATE-PCR samples similar to the one described below. Oncethe DNA mixture was prepared, we set up replicate LATE-PCR reactionscontaining either 100% heterozygous DNA or 90% heterozygous+10%homozygous DNA. Each LATE-PCR sample consisted of 1× Platinum Taq Buffer(Invitrogen, Carlsbad, Calif.), 3 mM MgCl₂, 250 μM dNTP mix, 0.24×SyberGold I (Invitrogen, Carlsbad, Calif.), 200 nM mispriming preventionreagent that we call Elixir compound 9-3iDD, 1.25 units Platinum Taqpolymerase (Invitrogen, Carlsbad, Calif.), 1 μM rs858521 Excess Primer,50 nM rs858521 Limiting primer, and 2.4 TM resonsense probe against thers858521 SNP G allele, and 1800 genome equivalent of the appropriategenomic DNA in a final volume of 25 μl. The sequence of the rs858521Excess Primer is

(SEQ. ID. No. 29) 5′ CAATCCCTTGACCTGTTGTGGAGAGAA 3′

The sequence of the rs858521 limiting primer is

(SEQ. ID. No. 30) 5′ TCCCCAGAGCCCAGCCGGTGTCATTTTC 3′

The sequence of the resonsense probe against the rs858521 SNP G alleleis

(SEQ. ID. No. 31) 5′ [Cy5] CTTCAGCTCAAA C AATA [Phos]

The sequence of the mispriming prevention reagent is 5′ Dabcyl

(SEQ. ID. No. 32) 5′ Dabcyl-CGCTATAATGAAATTATAGCG-Dabcyl

These samples were subjected to amplification in an ABI 7700 using athermal cycle profile consisting of one cycle of 95° C. for 3 min,followed by 45 cycles of 95° C. for 10 sec., 66° C. for 10 sec. and 72°C. for 20 sec. At the end of the reaction the reaction was melted from95° C. to 25° C. at 1° C. intervals for 1 min. at each temperature withfluorescence acquisition in the Cy5 channel. The clipped Cy5fluorescence signals with no baseline correction were exported into theExcel computer program. Calculation of the first derivative of thefluorescence signals was performed by subtracting the fluorescencesignals from one temperature from the fluorescence signals of the nexttemperature during the melt. Results are shown in FIG. 23, panels A andB. FIG. 23A shows the plot of the first derivative of fluorescencesignals versus temperature, that is, melting curves. The melting curvesin FIG. 23A were smoothed using the moving average function of Excel toeliminate the noise due to thermal fluctuations in the ABI 7700. FIG.23A revealed the melting peaks corresponding to the binding of the probeto its matched G allele target at higher temperatures and to themismatched C allele target at lower temperatures. FIG. 23A shows thatthe 90% heterozygous+10% homozygous samples, circle 231, exhibit a lowerG allele peak and a higher C allele melting peak relative to the heightsof the C allele and the G allele melting peaks in the 100% heterozygoussamples, circle 232. These differences are in accord with the expectedhigher proportion of the C allele in the 90% heterozygous+10% homozygoussample (55% C allele: 45% G allele) compared to the 100% heterozygoussample (50% G allele: 50% C allele). The ratio of the height of the Callele peak to the height of the G allele peak is shown as a bar graphin FIG. 23B. The set of bars on the right are for the 90%heterozygous+10% homozygous samples, corresponding to circle 231. Thedarker bars on the left are for the 100% heterozygous samples.Conventional error boxes 233 and 234 are shown for bar sets,respectively. This ratio distinguishes 100% heterozygous samples from90% heterozygous+10% homozygous samples with 99.7% certainty based onthe lack of overlap of the error boxes reflecting three standarddeviations of the error of the mean.

Example 15. Sensitivity of LATE-PCR Reactions to the Initial PolymeraseConcentration

PCR amplifications were performed utilizing an ABI 7700 to amplify the549 base amplicon designated as HVI-H within the d-loop region of humanmitochondrial DNA. Reaction Mixtures for genomic human DNA,Oligonucleotide Sequences (HV1-H), and LATE-PCR amplifications were asdescribed in Example 11, except the Units of Platinum Taq DNA polymerasevaried among samples, as follows: 0.125, 0.250, 0.375, 0.50, 0.625, and1.25 Units.

Melt curve analysis (SYBR green fluorescence versus temperature) wereperformed. Melt curves showed how the concentration of Taq influencedthe specificity of dsDNA product for this LATE-PCR reaction. As PlatinumTaq, concentration decreased from 1.25 units to 0.375 units thespecificity of the reaction increased, as reflected in the melting peaksof replicates. Lowering the concentration further, to 0.250 units,decreased specificity. At 0.125 units the reaction did not occur. Thegreatest specificity occurred with a Taq concentration of 0.375 units.

Example 16. Slope Variation as a Function of Taq Concentration in aReal-Time LATE-PCR and in a Real-Time Duplex LATE-PCR

We designed a duplex real-time LATE-PCR assay for simultaneousamplification of sequences within exons of the murine Oct4 and Xistgenes (GenBank Accession Number NM 013633 and L04961, respectively).Each reaction was run in a final volume of 50 μl and contained thefollowing reagents: 1×PCR buffer (Invitrogen, Carlsbad, Calif.)comprised by 20 mM Tris-HCl, pH 8.4, and 50 mM KCl, 3 mM MgCl₂, 0.4 mMof each dNTP, 50 nM Oct4 Limiting Primer having the sequence 5′TGGCTGGACACCTGGCTTCAGACT 3′ (SEQ ID NO: 33), 2 μM Oct4 Excess Primerhaving the sequence 5′ CAACTTGGGGGACTAGGC 3′ (SEQ ID NO: 34), 100 nMXist Limiting Primer having the sequence 5′ GGTCGTACAGGAAAAGATGGCGGCTCAA3′ (SEQ ID NO: 35), 2 μM Xist Excess Primer having the sequence 5′TGAAAGAAACCACTAGAGGGCA 3′ (SEQ ID NO:36), 1 μμM of a low melting-pointOct4 molecular beacon probe having the sequence 5′ TET-CCG CCT GGG ATGGCA TAC TGT GGA AGG CGG-Dabcyl 3′ (SEQ ID NO: 37) and 300 nM of amispriming prevention reagent (that we refer to as compound 9-3bDD)having the sequence 5′Dabcyl-CGTTATAATGAAATTATAACG-Dabcyl 3′ (SEQ. ID.No. 38). Antibody-complexed Platinum® Taq DNA polymerase (Invitrogen,Carlsbad, Calif.) was also included in the PCR mixture at concentrationsof 1, 2, or 3 Units per assay). A molecular beacon probe for thedetection of Xist amplicons was not added in this example.

In parallel with these duplex LATE-PCRs, we also ran a series of assaysfor LATE-PCR amplification of the Oct4 amplicon only. These assays hadidentical composition as the aforementioned duplexes, except for theomission of the Xist Limiting Primer and the Xist Excess Primer.

Mouse genomic DNA (Sigma, St Louis, Mo.) was added to all the assays andprovided the templates for PCR amplification. The number of genomesadded to each tube was calculated as 1000, based on a 6 pg/genome size(see Vendrely and Vendrely (1949) Experientia 5: 327-329).

All assays were run in duplicates. Amplification was carried out in anABI Prism 7700 Sequence Detector (Applied Biosystems, CA) with a thermalcycling profile comprised of 1 cycle at 95° C. for 5 minutes; 6 cyclesat 95° C. for 10 sec, 63° C. for 20 sec, and 72° C. for 30 sec; and 54cycles at 95° C. for 15 sec, 55° C. for 25 sec, 72° C. for 35 sec, and45° C. for 30 sec, with fluorescence acquisition at 45° C. in the TETchannel.

The results of this experiment are shown in FIG. 24, which plots thefluorescent signals generated by accumulating Oct4 amplicons throughhybridization with the TET-Oct4 molecular beacon probe. When only onepair of primers was present, increasing Taq polymerase concentrationfrom 1 Unit/assay (circle 241) to 2 Units/assay (circles 242) or 3Units/assay (circles 243) had the effect of making the slope of thesignals steeper, due to increased amplification efficiency. Signalsidentified by Circles 242 and 243 (2 and 3 Units/assay, respectively)were interspersed, suggesting that maximal efficiency had been reachedat approximately these levels. As expected, the slopes of the linesgenerated by the duplex reactions (circles 244, 245 and 246) were in allcases lower than those generated by amplification of a single amplicon,because the Taq polymerase was used at twice that rate. As in the caseof the single-amplicon LATE-PCR, augmenting Taq concentration in theduplex reaction from 1 Unit/assay (circle 244) to 2 Units/assay (circle245) or 3 Units/assay (circle 246) resulted in an increase in signalslope. There was no further increase in the initial slope of the 3Units/assay (circle 246) when compared to the initial slope of the 2Units/assay (circle 245), again suggesting that maximal efficiency hadbeen reached. However, the 3 Units/assay samples (circle 246) quicklyreached a plateau and the slope started declining, unlike that one ofthe 2 Units/assay samples (circle 245), indicating the probableoccurrence of mispriming in the presence of the highest Taqconcentration tested, which was not the case for samples 243, alsocontaining 3 Taq Units/assay but only one pair of primers. In spite ofthe higher amount of available Taq in the single-amplicon assays whencompared to the duplexes (3 units being used to generate one ampliconrather than two amplicons at the same time), more mispriming occurred inthe duplexes due to the addition of the Xist primers. In order to obtainmaximal efficiency without mispriming, Taq polymerase concentrationneeds, thus, to be optimized in consideration of the number andsequences of the primers added to the reaction.

What is claimed is:
 1. A sequential DNA amplification-sequencing methodcomprising: a) amplifying at least two DNA targets by LATE-PCR togenerate an amplification product containing copies of at least twoexcess primer strands and at least two limiting primer strands; b)processing the amplification product with a clean-up procedureconsisting of diluting the amplification product by a factor of at leasteighty to produce a cleaned-up amplification product; and c) sequencingthe copies of at least one of said excess primer strands in thecleaned-up amplification product.
 2. The method of claim 1, whereinsequencing is dideoxy cycle sequencing.
 3. The method of claim 1,wherein the act of diluting is performed in two steps.
 4. The method ofclaim 1, wherein the LATE-PCR comprises monitoring the adequacy ofproduction of single-stranded product, wherein the monitoring comprisesdetermining the ratio of single-stranded product to double-strandedproduct.
 5. The method of claim 1, wherein the excess primer strand issequenced using a primer having a sequence identical to a limitingprimer used in the LATE-PCR.